Hypersensitive response elicitor from Erwinia amylovora, its use, and encoding gene

ABSTRACT

The present invention is directed to an isolated protein or polypeptide which elicits a hypersensitive response in plants as well as an isolated DNA molecule which encodes the hypersensitive response eliciting protein or polypeptide. This isolated protein or polypeptide and the isolated DNA molecule can used to impart disease resistance to plants, to enhance plant growth, and/or to control insects on plants. This can be achieved by applying the hypersensitive response elicitor protein or polypeptide in a non-infectious form to plants or plant seeds under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds. Alternatively, transgenic plants or plant seeds transformed with a DNA molecule encoding a hypersensitive response elicitor protein or polypeptide can be provided and the transgenic plants or plants resulting from the transgenic plant seeds are grown under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds.

This application is a division of U.S. patent application Ser. No.09/120,663, filed Jul. 22, 1998, U.S. Pat. No. 6,228,644 and claimsbenefit of U.S. Provisional Patent Application Ser. No. 60/055,106,filed Aug. 4, 1997.

FIELD OF THE INVENTION

The present invention relates to a hypersensitive response elicitor fromErwinia amylovora, its use, and encoding gene.

BACKGROUND OF THE INVENTION

Interactions between bacterial pathogens and their plant hosts generallyfall into two categories: (1) compatible (pathogen-host), leading tointercellular bacterial growth, symptom development, and diseasedevelopment in the host plant; and (2) incompatible (pathogen-nonhost),resulting in the hypersensitive response, a particular type ofincompatible interaction occurring, without progressive diseasesymptoms. During compatible interactions on host plants, bacterialpopulations increase dramatically and progressive symptoms occur. Duringincompatible interactions, bacterial populations do not increase, andprogressive symptoms do not occur.

The hypersensitive response (“HR”) is a rapid, localized necrosis thatis associated with the active defense of plants against many pathogens(Kiraly, Z., “Defenses Triggered by the Invader: Hypersensitivity,”pages 201-224 in: Plant Disease: An Advanced Treatise, Vol. 5, J. G.Horsfall and E. B. Cowling, ed. Academic Press New York (1980); Klement,Z., “Hypersensitivity,” pages 149-177 in: Phytopathogenic Prokaryotes,Vol. 2, M. S. Mount and G. H. Lacy, ed. Academic Press, New York(1982)). The hypersensitive response elicited by bacteria is readilyobserved as a tissue collapse if high concentrations (≧10⁷ cells/ml) ofa limited host-range pathogen like Pseudomonas syringae or Erwiniaamylovora are infiltrated into the leaves of nonhost plants (necrosisoccurs only in isolated plant cells at lower levels of inoculum)(Klement, Z., “Rapid Detection of Pathogenicity of PhytopathogenicPseudomonads,” Nature 199:299-300; Klement, et al., “HypersensitiveReaction Induced by Phytopathogenic Bacteria in the Tobacco Leaf,”Phytopathology 54:474-477 (1963); Turner, et al., “The QuantitativeRelation Between Plant and Bacterial Cells Involved in theHypersensitive Reaction,” Phytopatholoy 64:885-890 (1974); Klement, Z.,“Hypersensitivity,” pages 149-177 in Phytopathogenic Prokaryotes, Vol.2., M. S. Mount and G. H. Lacy, ed. Academic Press, New York (1982)).The capacities to elicit the hypersensitive response in a nonhost and bepathogenic in a host appear linked. As noted by Klement, Z.,“Hypersensitivity,” pages 149-177 in Phytopathogenic Prokaryotes, Vol.2., M. S. Mount and G. H. Lacy, ed. Academic Press, New York, thesepathogens also cause physiologically similar, albeit delayed, necrosesin their interactions with compatible hosts. Furthermore, the ability toproduce the hypersensitive response or pathogenesis is dependent on acommon set of genes, denoted hrp (Lindgren, P. B., et al., “Gene Clusterof Pseudomonas syringae pv. ‘phaseolicola’ Controls Pathogenicity ofBean Plants and Hypersensitivity on Nonhost Plants,” J. Bacteriol.168:512-22 (1986); Willis, D. K., et al., “hrp Genes of PhytopathogenicBacteria,” Mol. Plant-Microbe Interact. 4:132-138 (1991)). Consequently,the hypersensitive response may hold clues to both the nature of plantdefense and the basis for bacterial pathogenicity.

The hrp genes are widespread in gram-negative plant pathogens, wherethey are clustered, conserved, and in some cases interchangeable(Willis, D. K., et al., “hrp Genes of Phytopathogenic Bacteria,” Mol.Plant-Microbe Interact. 4:132-138 (1991); Bonas, U., “hrp Genes ofPhytopathogenic Bacteria,” pages 79-98 in: Current Topics inMicrobiology and Immunology: Bacterial Pathogenesis of Plants andAnimals—Molecular and Cellular Mechanisms, J. L. Dangl, ed.Springer-Verlag, Berlin (1994)). Several hrp genes encode components ofa protein secretion pathway similar to one used by Yersinia, Shigella,and Salmonella spp. to secrete proteins essential in animal diseases(Van Gijsegem, et al., “Evolutionary Conservation of PathogenicityDeterminants Among Plant and Animal Pathogenic Bacteria,” TrendsMicrobiol. 1:175-180 (1993)). In E. amylovora, P. syringae, and P.solanacearum, hrp genes have been shown to control the production andsecretion of glycine-rich, protein elicitors of the hypersensitiveresponse (He, S. Y., et al. “Pseudomonas Syringae pv. SyringaeHarpin_(Pss): a Protein that is Secreted via the Hrp Pathway and Elicitsthe Hypersensitive Response in Plants,” Cell 73:1255-1266 (1993), Wei,Z.-H., et al., “HrpI of Erwinia amylovora Functions in Secretion ofHarpin and is a Member of a New Protein Family,” J. Bacteriol.175:7958-7967 (1993); Arlat, M. et al. “PopA1, a Protein Which Induces aHypersensitive-like Response on Specific Petunia Genotypes, is Secretedvia the Hrp Pathway of Pseudomonas solanacearum,” EMBO J. 13:543-553(1994)).

The first of these proteins was discovered in E. amylovora Ea321, abacterium that causes fire blight of rosaceous plants, and wasdesignated harpin (Wei, Z.-M., et al, “Harpin, Elicitor of theHypersensitive Response Produced by the Plant Pathogen Erwiniaamylovora,” Science 257:85-88 (1992)). Mutations in the encoding hrpNgene revealed that the hypersensitive response elicitor is required forE. amylovora to elicit a hypersensitive response in nonhost tobaccoleaves and incite disease symptoms in highly susceptible pear fruit. TheP. solanacearum GMI1000 PopA1 protein has similar physical propertiesand also elicits the hypersensitive response in leaves of tobacco, whichis not a host of that strain (Arlat, et al. “PopA1, a Protein WhichInduces a Hypersensitive-like Response on Specific Petunia Genotypes, isSecreted via the Hrp Pathway of Pseudomonas solanacearum,” EMBO J.13:543-53 (1994)). However, P. solanacearum popA mutants still elicitthe hypersensitive response in tobacco and incite disease in tomato.Thus, the role of these glycine-rich hypersensitive response elicitorscan vary widely among gram-negative plant pathogens.

Other plant pathogenic hypersensitive response elicitors have beenisolated and their encoding genes have been cloned and sequenced. Theseinclude: Erwinia chrysanthemi (Bauer, et. al., “Erwinia chrysanthemiHarpin_(Ech): Soft-Rot Pathogenesis,” MPMI 8(4): 484-91 (1995)); Erwiniacarotovora (Cui, et. al., “The RsmA Mutants of Erwinia carotovora subsp.carotovora Strain Ecc71 Overexpress hrPN_(ECC) and Elicit aHypersensitive Reaction-like Response in Tobacco Leaves,” MPMI 9(7):565-73 (1966)); Erwinia stewartii (Ahmad, et. al., “Harpin is notNecessary for the Pathogenicity of Erwinia stewartii on Maize,” 8thInt'l. Cong. Molec. Plant-Microb. Inter. Jul. 14-19, 1996 and Ahmad, et.al., “Harpin is not Necessary for the Pathogenicity of Erwinia stewartiion Maize,” Ann. Mtg. Am. Phytopath. Soc. Jul. 27-31, 1996); andPseudomonas syringae pv. syringae (WO 94/26782 to Cornell ResearchFoundation, Inc.).

The present invention is a further advance in the effort to identify,clone, and sequence hypersensitive response elicitor proteins orpolypeptides from plant pathogens.

SUMMARY OF THE INVENTION

The present invention is directed to an isolated protein or polypeptidewhich elicits a hypersensitive response in plants as well as an isolatedDNA molecule which encodes the hypersensitive response eliciting proteinor polypeptide.

The hypersensitive response eliciting protein or polypeptide can be usedto impart disease resistance to plants, to enhance plant growth, and/orto control insects. This involves applying the hypersensitive responseelicitor protein or polypeptide in a non-infectious form to plants orplant seeds under conditions effective to impart disease resistance, toenhance plant growth, and/or to control insects on plants or plantsgrown from the plant seeds.

As an alternative to applying the hypersensitive response elicitorprotein or polypeptide to plants or plant seeds in order to impartdisease resistance, to enhance plant growth, and/or to control insectson plants, transgenic plants or plant seeds can be utilized. Whenutilizing transgenic plants, this involves providing a transgenic planttransformed with a DNA molecule encoding a hypersensitive responseelicitor protein or polypeptide and growing the plant under conditionseffective to impart disease resistance, to enhance plant growth, and/orto control insects in the plants or plants grown from the plant seeds.Alternatively, a transgenic plant seed transformed with the DNA moleculeencoding a hypersensitive response elicitor protein or polypeptide canbe provided and planted in soil. A plant is then propagated underconditions effective to impart disease resistance, to enhance plantgrowth, and/or to control insects on plants or plants grown from theplant seeds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D show mutagenesis, complementation and heterologous expressionconstructs, and homology with and complementation of mutants by the avrElocus of P. syringae for the dspE operon of E. amylovora. Dashed boxesare uncharacterized ORFs; a filled triangle indicates a hrp (i.e.hypersensitive response elicitor encoding gene); box is a regulatorysequence that preceeds many hrp genes; and an open triangle indicatesanother promoter. Thick lines delineate the DNA for which sequence wasaccessioned. FIG. 1A shows the dsp/hrp gene cluster of E. amylovora inpCPP430. Operon names and types of proteins encoded are indicated at thetop. B, BamHI; E, EcoRI; H, HindIII. Half-arrows indicate internalpromoters without similarity to the hrp box consensus. FIG. 1B shows theregion downstream of hrpN containing the dspE operon. Circles markdeletion mutations and representative transposon insertions: black,non-pathogenic and HR⁺(i.e. hypersensitive response eliciting) or HRreduced (dsp); gray, reduced virulence and HR; white, wild-type. T104lies within the area marked by the dashed double arrow. K, Tn5miniKm; P,Tn5phoA; T, Tn10tet^(r); Δ, deletion mutation. The gray box is A/T-richDNA. FIG. 1C shows the clones and subclones of the dspE operon. Plasmiddesignations are indicated at the left, and vector-borne promoters atthe right. Restriction sites used for subcloning not shown above areshown in parentheses. A “+” aligned with a circle representing amutation in B indicates that the subclone complements that mutation.FIG. 1D shows the avrE locus (transcription units III and IV) of P.syringae pv. tomato DC3000 in pCPP2357. Percent amino acid identity ofthe predicted proteins AvrE and AvrF to DspE and DspF, respectively, areindicated. Black rectangles are transcriptional terminators (invertedrepeats). Complementation of mutations shown in FIG. 1B are depicted asin FIG. 1C, above.

FIG. 2 shows the expression of the full-length and the N-terminal halfof DspE in recombinant E. coli DH5α. Lysates of cells carrying eitherpCPP1259, containing the entire dspE operon (lane A); pCPP50, thecloning vector (lane B); or pCPP1244, containing only the 5′ half of thedspE gene (lane C), were subjected to SDS-PAGE followed by Coomassiestaining. Bands corresponding to DspE (lane A) and the N-terminal halfof DspE (lane C) are marked by arrows. Migration of molecular weightmarkers is indicated on the left.

FIGS. 3A-D show the role of dspe in pathogenicity and HR elicitation.FIG. 3A shows immature pear fruit 4 days after inoculation with (left toright) strains Ea321, Ea321dspEΔ1554, or Ea321dspEΔ1554 harboring the 5′half of dspE on pCPP1237. FIG. 3B shows Norchief soybean leaf 24 hrafter infiltration with (1) Ea321, (2) Ea321dspEΔ1554, (3) Ea321hrpN::Tn5 (Wei, et al., Science, 257:85-88 (1992), which is herebyincorporated by reference), and (4) Ea321 hrpL::Tn5 (Wei, et al., J.Bacteriol., 177:6201-10 (1995), which is hereby incorporated byreference). FIG. 3C shows a tobacco leaf 48 hr after infiltration withparallel dilution series of suspensions of strains (left) Ea321 and(right) Ea321dspEA1554. The concentrations infiltrated (top to bottom)are 1×10¹⁰, 1×10⁹, 5×10⁸, and 5×10⁷ cfu/ml. FIG. 3D is like FIG. 3Cexcept the more virulent strain Ea273 and corresponding mutantEa273dspEΔ1554 were used, and concentrations ranged from 5×10⁹ to 5×10⁵cfu/ml in log increments.

FIG. 4 shows the expression of a promoterless GUS construct fused todspE in E. amylovora Ea273. Ea273 and Ea273dspE::uidA (a merodiploidcontaining both a wild-type dspe and a truncated dspE fused to the uidAgene; black bars) were grown in LB or Hrp MM, or inoculated to immaturepear fruit. Ea273dspE::uidAhrpL::Tn5 (dark gray bar) and Ea273hrcV::TnSuidA (light gray bar) were also grown in hrp MM. Values shownrepresent means of triplicate samples normalized for bacterial cellconcentration. Standard deviations are represented by lines extendingfrom each bar. The mean values for three samples of Ea273 in each assaywere subtracted from, and standard deviations added to, thecorresponding values obtained for the other strains.

FIGS. 5A-C show the transgeneric avirulence function of the dspE operonand complementation of a dspE mutant with the avrE locus. Norchiefsoybean leaves were either (See FIG. 5A) infiltrated with 1×10⁸ cfu/mlsuspensions of (left) P. syringae pv. glycinea race 4 carrying pCPP1250(containing the dspE operon) or (right) pML 122 (the cloning vector) andphotographed after 24 hr at room temperature or (See FIG. 5B)infiltrated with 8×10⁵ cfu/ml suspensions of the same strains andphotographed after seven days at 22° C. and high relative humidity.Tissue collapse is apparent on both leaves where the strain carryingpCPP1250 was infiltrated. On the leaf incubated for seven days,chlorosis extending beyond the infiltrated area, typical of disease, isapparent on the half infiltrated with the strain carrying the vectoronly. The dark section on the side of the leaf infiltrated with thestrain carrying pCPP1250 is a shadow caused by a buckle in the leaf.FIG. 5C shows pear halves inoculated with (left to right) Ea321,Ea321dspEΔ1521(pCPP2357, containing the avrE locus), orEa321dspEΔ1521(pCPP2357avrE::Tn5uidA) and photographed after seven days.Although symptoms are greatly reduced relative to wild type, necrosis isapparent around and drops of ooze can be seen within the well of thefruit inoculated with the dspE strain carrying the intact avrE locus.The fruit inoculated with the dspE strain carrying a disrupted clone ofavrE is symptomless.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an isolated DNA molecule having anucleotide sequence of SEQ. ID. No. 1 as follows:

ATGGAATTAA AATCACTGGG AACTGAACAC AAGGCGGCAG TACACACAGC GGCGCACAAC 60CCTGTGGGGC ATGGTGTTGC CTTACAGCAG GGCAGCAGCA GCAGCAGCCC GCAAAATGCC 120GCTGCATCAT TGGCGGCAGA AGGCAAAAAT CGTGGGAAAA TGCCGAGAAT TCACCAGCCA 180TCTACTGCGG CTGATGGTAT CAGCGCTGCT CACCAGCAAA AGAAATCCTT CAGTCTCAGG 240GGCTGTTTGG GGACGAAAAA ATTTTCCAGA TCGGCACCGC AGGGCCAGCC AGGTACCACC 300CACAGCAAAG GGGCAACATT GCGCGATCTG CTGGCGCGGG ACGACGGCGA AACGCAGCAT 360GAGGCGGCCG CGCCAGATGC GGCGCGTTTG ACCCGTTCGG GCGGCGTCAA ACGCCGCAAT 420ATGGACGACA TGGCCGGGCG GCCAATGGTG AAAGGTGGCA GCGGCGAAGA TAAGGTACCA 480ACGCAGCAAA AACGGCATCA GCTGAACAAT TTTGGCCAGA TGCGCCAAAC GATGTTGAGC 540AAAATGGCTC ACCCGGCTTC AGCCAACGCC GGCGATCGCC TGCAGCATTC ACCGCCGCAC 600ATCCCGGGTA GCCACCACGA AATCAAGGAA GAACCGGTTG GCTCCACCAG CAAGGCAACA 660ACGGCCCACG CAGACAGAGT GGAAATCGCT CAGGAAGATG ACGACAGCGA ATTCCAGCAA 720CTGCATCAAC AGCGGCTGGC GCGCGAACGG GAAAATCCAC CGCAGCCGCC CAAACTCGGC 780GTTGCCACAC CGATTAGCGC CAGGTTTCAG CCCAAACTGA CTGCGGTTGC GGAAAGCGTC 840CTTGAGGGGA CAGATACCAC GCAGTCACCC CTTAAGCCGC AATCAATGCT GAAAGGAAGT 900GGAGCCGGGG TAACGCCGCT GGCGGTAACG CTGGATAAAG GCAAGTTGCA GCTGGCACCG 960GATAATCCAC CCGCGCTCAA TACGTTGTTG AAGCAGACAT TGGGTAAAGA CACCCAGCAC 1020TATCTGGCGC ACCATGCCAG CAGCGACGGT AGCCAGCATC TGCTGCTGGA CAACAAAGGC 1080CACCTGTTTG ATATCAAAAG CACCGCCACC AGCTATAGCG TGCTGCACAA CAGCCACCCC 1140GGTGAGATAA AGGGCAAGCT GGCGCAGGCG GGTACTGGCT CCGTCAGCGT AGACGGTAAA 1200AGCGGCAAGA TCTCGCTGGG GAGCGGTACG CAAAGTCACA ACAAAACAAT GCTAAGCCAA 1260CCGGGGGAAG CGCACCGTTC CTTATTAACC GGCATTTGGC AGCATCCTGC TGGCGCAGCG 1320CGGCCGCAGG GCGAGTCAAT CCGCCTGCAT GACGACAAAA TTCATATCCT GCATCCGGAG 1380CTGGGCGTAT GGCAATCTGC GGATAAAGAT ACCCACAGCC AGCTGTCTCG CCAGGCAGAC 1440GGTAACCTCT ATGCGCTTAA AGACAACCGT ACCCTGCAAA ACCTCTCCGA TAATAAATCC 1500TCAGAAAAGC TCCTCGATAA AATCAAATCG TATTCCGTTG ATCAGCGGGG GCAGGTGGCG 1560ATCCTGACGG ATACTCCCGG CCGCCATAAC ATGAGTATTA TGCCCTCGCT GGATGCTTCC 1620CCGGAGAGCC ATATTTCCCT CAGCCTGCAT TTTGCCGATG CCCACCAGGG GTTATTGCAC 1680GGGAACTCGG AGCTTGAGGC ACAATCTGTC GCGATCAGCC ATGGGCGACT GGTTGTGGCC 1740GATAGCGAAG GCAAGCTGTT TAGCGCCGCC ATTCCGAACC AAGGCGATGG AAACGAACTG 1800AAAATGAAAG CCATGCCTCA GCATGCGCTC GATGAACATT TTGGTCATGA CCACCAGATT 1860TCTGGATTTT TCCATGACGA CCACGGCCAG CTTAATGCGC TGGTGAAAAA TAACTTCAGG 1920CAGCAGCATG CCTGCCCGTT GGGTAACGAT CATCAGTTTC ACCCCGGCTG GAACCTGACT 1960GATGCGCTGG TTATCGACAA TCAGCTCGGG CTGCATCATA CCAATCCTGA ACCGCATGAG 2040ATTCTTGATA TGGCGCATTT AGGCAGCCTG GCGTTACAGG AGGGCAAGCT TCACTATTTT 2100GACCAGCTGA CCAAAGGGTG GACTGGCCCG GAGTCAGATT CTAAGCAGCT GAAAAAAGGC 2160CTGGATGGAG CAGCTTATCT ACTGAAAGAC GGTGAAGTGA AACGCCTGAA TATTAATCAG 2220AGCACCTCCT CTATCAAGCA CGGAACGGAA AACGTTTTTT CGCTGCCGCA TGTGCGCAAT 2280AAACCGGAGC CGGGACATGC CCTGCAAGGG CTGAATAAAG ACGATAACGC CCAGGCCATG 2340GCGGTGATTG GGGTAAATAA ATACCTGGCG CTGACGGAAA AAGGGGACAT TCGCTCCTTC 2400CAGATAAAAC CCGGCACCCA GCAGTAACAG CGGCCGGCAC AAACTCTCAG CCGCGAAGGT 2460ATCAGCGGCG AACTCAAAGA CATTCATGTC GACCACAAGC AGAACCTGTA TGCCTTGACC 2520CACGAGGGAG AGGTGTTTCA TCAGCCGCGT GAAGCCTGGC AGAATGGTGC CGAAAGCAGC 2580AGCTGGCACA AACTGGCGTT GCCACAGAGT GAAAGTAAGC TAAAAAGTCT GGACATGAGC 2640CATGAGCACA AACCGATTGC CACCTTTGAA GACGGTAGCC AGCATCAGCT GAAGGCTGGC 2700GGCTGGCACG CCTATGCGGC ACCTGAACGC GGGCCGCTGG CGGTGGGTAC CAGCGGTTCA 2760CAAACCGTCT TTAACCGACT AATGCAGGGG GTGAAAGGCA AGGTGATCCC AGGCAGCGGG 2820TTGACGGTTA AGCTCTCCGC TCAGACGGGG GGAATGACCG GCGCCGAACG GCGCAAGGTC 2680AGCAGTAAAT TTTCCGAAAG GATCCGCGCC TATGCGTTCA ACCCAACAAT GTCCACGCCG 2940CGACCGATTA AAAATGCTGC TTATGCCACA CAGCACGGCT GGCAGGGGCG TGAGGGGTTG 3000AAGCCGTTGT ACGAGATGCA GGGAGCGCTG ATTAAACAAC TGGATGCGCA TAACGTTCGT 3060CATAACGCGC CACAGCCAGA TTTGCAGAGC AAACTGGAAA CTCTGGATTT AGGCGAACAT 3120GGCGCAGAAT TGCTTAACGA CATGAAGCGC TTCCGCGACG AACTGGAGCA GAGTGCAACC 3160CGTTCGGTGA CCGTTTTAGG TCAACATCAG GGAGTGCTAA AAAGCAACGG TGAAATCAAT 3240AGCGAATTTA AGCCATCGCC CGGCAAGGCG TTGGTCCAGA GCTTTAACGT CAATCGCTCT 3300GGTCAGGATC TAAGCAAGTC ACTGCAACAG GCAGTACATG CCACGCCGCC ATCCGCAGAG 3360AGTAAACTGC AATCCATGCT GGGGCACTTT GTCAGTGCCG GGGTGGATAT GAGTCATCAG 3420AAGGGCGAGA TCCCGCTGGG CCGCCAGCGC GATCCGAATG ATAAAACCGC ACTGACCAAA 3480TCGCGTTTAA TTTTAGATAC CGTGACCATC GGTGAACTGC ATGAACTGGC CGATAAGGCG 3540AAACTGGTAT CTGACCATAA ACCCGATGCC GATCAGATAA AACAGCTGCG CCAGCACTTC 3600GATACGCTGC GTGAAAAGCG GTATGAGAGC AATCCGGTGA AGCATTACAC CGATATGGGC 3660TTCACCCATA ATAAGGCGCT GGAAGCAAAC TATGATGCGG TCAAAGCCTT TATCAATGCC 3720TTTAAGAAAG AGCACCACGG CGTCAATCTG ACCACGCGTA CCGTACTGGA ATCACAGGGC 3780AGTGCGGAGC TGGCGAAGAA GCTCAAGAAT ACGCTGTTGT CCCTGGACAG TGGTGAAAGT 3840ATGAGCTTCA GCCGGTCATA TGGCGGGGGC GTCAGCACTG TCTTTGTGCC TACCCTTAGC 3900AAGAAGGTGC CAGTTCCGGT GATCCCCGGA GCCGGCATCA CGCTGGATCG CGCCTATAAC 3960CTGAGCTTCA GTCGTACCAG CGGCGGATTG AACGTCAGTT TTGGCCGCGA CGGCGGGGTG 4020AGTGGTAACA TCATGGTCGC TACCGGCCAT GATGTGATGC CCTATATGAC CGGTAAGAAA 4080ACCAGTGCAG GTAACGCCAG TGACTGGTTG AGCGCAAAAC ATAAAATCAG CCCGGACTTG 4140CGTATCGGCG CTGCTGTGAG TGGCACCCTG CAAGGAACGC TACAAAACAG CCTGAAGTTT 4200AAGCTGACAG AGGATGAGCT GCCTGGCTTT ATCCATGGCT TGACGCATGG CACGTTGACC 4260CCGGCAGAAC TGTTGCAAAA GGGGATCGAA CATCAGATGA AGCAGGGCAG CAAACTGACG 4320TTTAGCGTCG ATACCTCGGC AAATCTGGAT CTGCCTGCCG GTATCAATCT GAACGAAGAC 4380GGCAGTAAAC CAAATGGTGT CACTGCCCGT GTTTCTGCCG GGCTAAGTGC ATCGGCAAAC 4440CTGGCCGCCG GCTCGCGTGA ACGCAGCACC ACCTCTGGCC AGTTTGGCAG CACGACTTCG 4500GCCAGCAATA ACCGCCCAAC CTTCCTCAAC GGGGTCGGCG CGGGTGCTAA CCTGACGGCT 4560GCTTTAGGGG TTGCCCATTC ATCTACGCAT GAAGGGAAAC CGGTCGGGAT CTTCCCGGCA 4620TTTACCTCGA CCAATGTTTC GGCAGCGCTG GCGCTGGATA ACCGTACCTC ACAGAGTATC 4680AGCCTGGAAT TGAAGCGCGC GGAGCCGGTG ACCAGCAACG ATATCAGCGA GTTTACCTCC 4740ACGCTGGGAA AACACTTTAA GGATAGCGCC ACAACGAAGA TGCTTGCCGC TCTCAAAGAG 4800TTAGATGACG CTAAGCCCGC TGAACAACTG CATATTTTAC AGCAGCATTT CAGTGCAAAA 4860GATGTCGTCG GTGATGAACG CTACGAGGCG GTGCGCAACC TGAAAAAACT GGTGATACGT 4920CAACAGGCTG CGGACAGCCA CAGCATGGAA TTAGGATCTG CCAGTCACAG CACGACCTAC 4980AATAATCTGT CGAGAATAAA TAATGACGGC ATTGTCGAGC TGCTACACAA ACATTTCGAT 5040GCGGCATTAC CAGCAAGCAG TGCCAAACGT CTTGGTGAAA TGATGAATAA CGATCCGGCA 5100CTGAAAGATA TTATTAAGCA GCTGCAAAGT ACGCCGTTCA GCAGCGCCAG CGTGTCGATG 5160GAGCTGAAAG ATGGTCTGCG TGAGCAGACG GAAAAAGCAA TACTGGACGG TAAGGTCGGT 5220CGTGAAGAAG TGGGAGTACT TTTCCAGGAT CGTAACAACT TGCGTGTTAA ATCGGTCAGC 5280GTCAGTCAGT CCGTCAGCAA AAGCGAAGGC TTCAATACCC CAGCGCTGTT ACTGGGGACG 5340AGCAACAGCG CTGCTATGAG CATGGAGCGC AACATCGGAA CCATTAATTT TAAATACGGC 5400CAGGATCAGA ACACCCCACG GCGATTTACC CTGGAGGGTG GAATAGCTCA GGCTAATCCG 5460CAGGTCGCAT CTGCGCTTAC TGATTTGAAG AAGGAAGGGC TGGAAATGAA GAGCTAA 5517This DNA molecule is known as the dspE gene. This isolated DNA moleculeof the present invention encodes a protein or polypeptide which elicitsa plant pathogen's hypersensitive response having an amino acid sequenceof SEQ. ID. No. 2 as follows:

Met Glu Leu Lys Ser Leu Gly Thr Glu His Lys Ala Ala Val His Thr1               5                   10                  15 Ala Ala HisAsn Pro Val Gly His Gly Val Ala Leu Gln Gln Gly Ser            20                  25                  30 Ser Ser Ser SerPro Gln Asn Ala Ala Ala Ser Leu Ala Ala Glu Gly        35                  40                  45 Lys Asn Arg Gly LysMet Pro Arg Ile His Gln Pro Ser Thr Ala Ala    50                  55                  60 Asp Gly Ile Ser Ala AlaHis Gln Gln Lys Lys Ser Phe Ser Leu Arg65                  70                  75                  80 Gly CysLeu Gly Thr Lys Lys Phe Ser Arg Ser Ala Pro Gln Gly Gln                85                  90                  95 Pro Gly ThrThr His Ser Lys Gly Ala Thr Leu Arg Asp Leu Leu Ala            100                 105                 110 Arg Asp Asp GlyGlu Thr Gln His Glu Ala Ala Ala Pro Asp Ala Ala        115                 120                 125 Arg Leu Thr Arg SerGly Gly Val Lys Arg Arg Asn Met Asp Asp Met    130                 135                 140 Ala Gly Arg Pro Met ValLys Gly Gly Ser Gly Glu Asp Lys Val Pro145                 150                 155                 160 Thr GlnGln Lys Arg His Gln Leu Asn Asn Phe Gly Gln Met Arg Gln                165                 170                 175 Thr Met LeuSer Lys Met Ala His Pro Ala Ser Ala Asn Ala Gly Asp            180                 185                 190 Arg Leu Gln HisSer Pro Pro His Ile Pro Gly Ser His His Glu Ile        195                 200                 205 Lys Glu Glu Pro ValGly Ser Thr Ser Lys Ala Thr Thr Ala His Ala    210                 215                 220 Asp Arg Val Glu Ile AlaGln Glu Asp Asp Asp Ser Glu Phe Gln Gln225                 230                 235                 240 Leu HisGln Gln Arg Leu Ala Arg Glu Arg Glu Asn Pro Pro Gln Pro                245                 250                 255 Pro Lys LeuGly Val Ala Thr Pro Ile Ser Ala Arg Phe Gln Pro Lys            260                 265                 270 Leu Thr Ala ValAla Glu Ser Val Leu Glu Gly Thr Asp Thr Thr Gln        275                 280                 285 Ser Pro Leu Lys ProGln Ser Met Leu Lys Gly Ser Gly Ala Gly Val    290                 295                 300 Thr Pro Leu Ala Val ThrLeu Asp Lys Gly Lys Leu Gln Leu Ala Pro305                 310                 315                 320 Asp AsnPro Pro Ala Leu Asn Thr Leu Leu Lys Gln Thr Leu Gly Lys                325                 330                 335 Asp Thr GlnHis Tyr Leu Ala His His Ala Ser Ser Asp Gly Ser Gln            340                 345                 350 His Leu Leu LeuAsp Asn Lys Gly His Leu Phe Asp Ile Lys Ser Thr        355                 360                 365 Ala Thr Ser Tyr SerVal Leu His Asn Ser His Pro Gly Glu Ile Lys    370                 375                 380 Gly Lys Leu Ala Gln AlaGly Thr Gly Ser Val Ser Val Asp Gly Lys385                 390                 395                 400 Ser GlyLys Ile Ser Leu Gly Ser Gly Thr Gln Ser His Asn Lys Thr                405                 410                 415 Met Leu SerGln Pro Gly Glu Ala His Arg Ser Leu Leu Thr Gly Ile            420                 425                 430 Trp Gln His ProAla Gly Ala Ala Arg Pro Gln Gly Glu Ser Ile Arg        435                 440                 445 Leu His Asp Asp LysIle His Ile Leu His Pro Glu Leu Gly Val Trp    450                 455                 460 Gln Ser Ala Asp Lys AspThr His Ser Gln Leu Ser Arg Gln Ala Asp465                 470                 475                 480 Gly LysLeu Tyr Ala Leu Lys Asp Asn Arg Thr Leu Gln Asn Leu Ser                485                 490                 495 Asp Asn LysSer Ser Glu Lys Leu Val Asp Lys Ile Lys Ser Tyr Ser            500                 505                 510 Val Asp Gln ArgGly Gln Val Ala Ile Leu Thr Asp Thr Pro Gly Arg        515                 520                 525 His Lys Met Ser IleMet Pro Ser Leu Asp Ala Ser Pro Glu Ser His    530                 535                 540 Ile Ser Leu Ser Leu HisPhe Ala Asp Ala His Gln Gly Leu Leu His545                 550                 555                 560 Gly LysSer Glu Leu Glu Ala Gln Ser Val Ala Ile Ser His Gly Arg                565                 570                 575 Leu Val ValAla Asp Ser Glu Gly Lys Leu Phe Ser Ala Ala Ile Pro            580                 585                 590 Lys Gln Gly AspGly Asn Glu Leu Lys Met Lys Ala Met Pro Gln His        595                 600                 605 Ala Leu Asp Glu HisPhe Gly His Asp His Gln Ile Ser Gly Phe Phe    610                 615                 620 His Asp Asp His Gly GlnLeu Asn Ala Leu Val Lys Asn Asn Phe Arg625                 630                 635                 640 Gln GlnHis Ala Cys Pro Leu Gly Asn Asp His Gln Phe His Pro Gly                645                 650                 655 Trp Asn LeuThr Asp Ala Leu Val Ile Asp Asn Gln Leu Gly Leu His            660                 665                 670 His Thr Asn ProGlu Pro His Glu Ile Leu Asp Met Gly His Leu Gly        675                 680                 685 Ser Leu Ala Leu GlnGlu Gly Lys Leu His Tyr Phe Asp Gln Leu Thr    690                 695                 700 Lys Gly Trp Thr Gly AlaGlu Ser Asp Cys Lys Gln Leu Lys Lys Gly705                 710                 715                 720 Leu AspGly Ala Ala Tyr Leu Leu Lys Asp Gly Glu Val Lys Arg Leu                725                 730                 735 Asn Ile AsnGln Ser Thr Ser Ser Ile Lys His Gly Thr Glu Asn Val            740                 745                 750 Phe Ser Leu ProHis Val Arg Asn Lys Pro Glu Pro Gly Asp Ala Leu        755                 760                 765 Gln Gly Leu Asn LysAsp Asp Lys Ala Gln Ala Met Ala Val Ile Gly    770                 775                 780 Val Asn Lys Tyr Leu AlaLeu Thr Glu Lys Gly Asp Ile Arg Ser Phe785                 790                 795                 800 Gln IleLys Pro Gly Thr Gln Gln Leu Glu Arg Pro Ala Gln Thr Leu                805                 810                 815 Ser Arg GluGly Ile Ser Gly Glu Leu Lys Asp Ile His Val Asp His            820                 825                 830 Lys Gln Asn LeuTyr Ala Leu Thr His Glu Gly Glu Val Phe His Gln        835                 840                 845 Pro Arg Glu Ala TrpGln Asn Gly Ala Glu Ser Ser Ser Trp His Lys    850                 855                 860 Leu Ala Leu Pro Gln SerGlu Ser Lys Leu Lys Ser Leu Asp Met Ser865                 870                 875                 880 His GluHis Lys Pro Ile Ala Thr Phe Glu Asp Gly Ser Gln His Gln                885                 890                 895 Leu Lys AlaGly Gly Trp His Ala Tyr Ala Ala Pro Glu Arg Gly Pro            900                 905                 910 Leu Ala Val GlyThr Ser Gly Ser Gln Thr Val Phe Asn Arg Leu Met        915                 920                 925 Gln Gly Val Lys GlyLys Val Ile Pro Gly Ser Gly Leu Thr Val Lys    930                 935                 940 Leu Ser Ala Gln Thr GlyGly Met Thr Gly Ala Glu Gly Arg Lys Val945                 950                 955                 960 Ser SerLys Phe Ser Glu Arg Ile Arg Ala Tyr Ala Phe Asn Pro Thr                965                 970                 975 Met Ser ThrPro Arg Pro Ile Lys Asn Ala Ala Tyr Ala Thr Gln His            980                 985                 990 Gly Trp Gln GlyArg Glu Gly Leu Lys Pro Leu Tyr Glu Met Gln Gly        995                 1000                1005 Ala Leu Ile Lys GlnLeu Asp Ala His Asn Val Arg His Asn Ala Pro    1010                1015                1020 Gln Pro Asp Leu Gln SerLys Leu Glu Thr Leu Asp Leu Gly Glu His1025                1030                1035                1040 Gly AlaGlu Leu Leu Asn Asp Met Lys Arg Phe Arg Asp Glu Leu Glu                1045                1050                1055 Gln Ser AlaThr Arg Ser Val Thr Val Leu Gly Gln His Gln Gly Val            1060                1065                1070 Leu Lys Ser AsnGly Glu Ile Asn Ser Glu Phe Lys Pro Ser Pro Gly        1075                1080                1085 Lys Ala Leu Val GlnSer Phe Asn Val Asn Arg Ser Gly Gln Asp Leu    1090                1095                1100 Ser Lys Ser Leu Gln GlnAla Val His Ala Thr Pro Pro Ser Ala Glu1105                1110                1115                1120 Ser LysLeu Gln Ser Met Leu Gly His Phe Val Ser Ala Gly Val Asp                1125                1130                1135 Met Ser HisGln Lys Gly Glu Ile Pro Leu Gly Arg Gln Arg Asp Pro            1140                1145                1150 Asn Asp Lys ThrAla Leu Thr Lys Ser Arg Leu Ile Leu Asp Thr Val        1155                1160                1165 Thr Ile Gly Glu LeuHis Glu Leu Ala Asp Lys Ala Lys Leu Val Ser    1170                1175                1180 Asp His Lys Pro Asp AlaAsp Gln Ile Lys Gln Leu Arg Gln Gln Phe1185                1190                1195                1200 Asp ThrLeu Arg Glu Lys Arg Tyr Glu Ser Asn Pro Val Lys His Tyr                1205                1210                1215 Thr Asp MetGly Phe Thr His Asn Lys Ala Leu Glu Ala Asn Tyr Asp            1220                1225                1230 Ala Val Lys AlaPhe Ile Asn Ala Phe Lys Lys Glu His His Gly Val        1235                1240                1245 Asn Leu Thr Thr ArgThr Val Leu Glu Ser Gln Gly Ser Ala Glu Leu    1250                1255                1260 Ala Lys Lys Leu Lys AsnThr Leu Leu Ser Leu Asp Ser Gly Glu Ser1265                1270                1275                1280 Met SerPhe Ser Arg Ser Tyr Gly Gly Gly Val Ser Thr Val Phe Val                1285                1290                1295 Pro Thr LeuSer Lys Lys Val Pro Val Pro Val Ile Pro Gly Ala Gly            1300                1305                1310 Ile Thr Leu AspArg Ala Tyr Asn Leu Ser Phe Ser Arg Thr Ser Gly        1315                1320                1325 Gly Leu Asn Val SerPhe Gly Arg Asp Gly Gly Val Ser Gly Asn Ile    1330                1335                1340 Met Val Ala Thr Gly HisAsp Val Met Pro Tyr Met Thr Gly Lys Lys1345                1350                1355                1360 Thr SerAla Gly Asn Ala Ser Asp Trp Leu Ser Ala Lys His Lys Ile                1365                1370                1375 Ser Pro AspLeu Arg Ile Gly Ala Ala Val Ser Gly Thr Leu Gln Gly            1380                1385                1390 Thr Leu Gln AsnSer Leu Lys Phe Lys Leu Thr Glu Asp Glu Leu Pro        1395                1400                1405 Gly Phe Ile His GlyLeu Thr His Gly Thr Leu Thr Pro Ala Glu Leu    1410                1415                1420 Leu Gln Lys Gly Ile GluHis Gln Met Lys Gln Gly Ser Lys Leu Thr1425                1430                1435                1440 Phe SerVal Asp Thr Ser Ala Asn Leu Asp Leu Arg Ala Gly Ile Asn                1445                1450                1455 Leu Asn GluAsp Gly Ser Lys Pro Asn Gly Val Thr Ala Arg Val Ser            1460                1465                1470 Ala Gly Leu SerAla Ser Ala Asn Leu Ala Ala Gly Ser Arg Glu Arg        1475                1480                1485 Ser Thr Thr Ser GlyGln Phe Gly Ser Thr Thr Ser Ala Ser Asn Asn    1490                1495                1500 Arg Pro Thr Phe Leu AsnGly Val Gly Ala Gly Ala Asn Leu Thr Ala1505                1510                1515                1520 Ala LeuGly Val Ala His Ser Ser Thr His Glu Gly Lys Pro Val Gly                1525                1530                1535 Ile Phe ProAla Phe Thr Ser Thr Asn Val Ser Ala Ala Leu Ala Leu            1540                1545                1550 Asp Asn Arg ThrSer Gln Ser Ile Ser Leu Glu Leu Lys Arg Ala Glu        1555                1560                1565 Pro Val Thr Ser AsnAsp Ile Ser Glu Leu Thr Ser Thr Leu Gly Lys    1570                1575                1580 His Phe Lys Asp Ser AlaThr Thr Lys Met Leu Ala Ala Leu Lys Glu1585                1590                1595                1600 Leu AspAsp Ala Lys Pro Ala Glu Gln Leu His Ile Leu Gln Gln His                1605                1610                1615 Phe Ser AlaLys Asp Val Val Gly Asp Glu Arg Tyr Glu Ala Val Arg            1620                1625                1630 Asn Leu Lys LysLeu Val Ile Arg Gln Gln Ala Ala Asp Ser His Ser        1635                1640                1645 Met Glu Leu Gly SerAla Ser His Ser Thr Thr Tyr Asn Asn Leu Ser    1650                1655                1660 Arg Ile Asn Asn Asp GlyIle Val Glu Leu Leu His Lys His Phe Asp1665                1670                1675                1680 Ala AlaLeu Pro Ala Ser Ser Ala Lys Arg Leu Gly Glu Met Met Asn                1685                1690                1695 Asn Asp ProAla Leu Lys Asp Ile Ile Lys Gln Leu Gln Ser Thr Pro            1700                1705                1710 Phe Ser Ser AlaSer Val Ser Met Glu Leu Lys Asp Gly Leu Arg Glu        1715                1720                1725 Gln Thr Glu Lys AlaIle Leu Asp Gly Lys Val Gly Arg Glu Glu Val    1730                1735                1740 Gly Val Leu Phe Gln AspArg Asn Asn Leu Arg Val Lys Ser Val Ser1745                1750                1755                1760 Val SerGln Ser Val Ser Lys Ser Glu Gly Phe Asn Thr Pro Ala Leu                1765                1770                1775 Leu Leu GlyThr Ser Asn Ser Ala Ala Met Ser Met Glu Arg Asn Ile            1780                1785                1790 Gly Thr Ile AsnPhe Lys Tyr Gly Gln Asp Gln Asn Thr Pro Arg Arg        1795                1800                1805 Phe Thr Leu Glu GlyGly Ile Ala Gln Ala Asn Pro Gln Val Ala Ser    1810                1815                1820 Ala Leu Thr Asp Leu LysLys Glu Gly Leu Glu Met Lys Ser1825                    1830            1835This protein or polypeptide is about 198 kDa and has a pI of 8.98.

The present invention relates to an isolated DNA molecule having anucleotide sequence of SEQ. ID. No. 3 as follows:

ATGACATCGT CACAGCAGCG GGTTGAAAGG TTTTTACAGT ATTTCTCCGC CGGGTGTAAA 60ACGCCCATAC ATCTGAAAGA CGGGGTGTGC GCCCTGTATA ACGAACAAGA TGAGGAGGCG 120GCGGTGCTGG AAGTACCGCA ACACAGCGAC AGCCTGTTAC TACACTGCCG AATCATTGAG 180GCTGACCCAC AAACTTCAAT AACCCTGTAT TCGATGCTAT TACAGCTGAA TTTTGAAATG 240GCGGCCATGC GCGGCTGTTG GCTGGCGCTG GATGAACTGC ACAACGTGCG TTTATGTTTT 300CAGCAGTCGC TGGAGCATCT GGATGAAGCA AGTTTTAGCG ATATCGTTAG CGGCTTCATC 360GAACATGCGG CAGAAGTGCG TGAGTATATA GCGCAATTAG ACGAGAGTAG CGCGGCATAA 420This is known as the dspF gene. This isolated DNA molecule of thepresent invention encodes a hypersensitive response elicitor protein orpolypeptide having an amino acid sequence of SEQ. ID. No. 4 as follows:

Met Thr Ser Ser Gln Gln Arg Val Glu Arg Phe Leu Gln Tyr Phe Ser1               5                   10                  15 Ala Gly CysLys Thr Pro Ile His Leu Lys Asp Gly Val Cys Ala Leu            20                  25                  30 Tyr Asn Glu GlnAsp Glu Glu Ala Ala Val Leu Glu Val Pro Gln His        35                  40                  45 Ser Asp Ser Leu LeuLeu His Cys Arg Ile Ile Glu Ala Asp Pro Gln    50                  55                  60 Thr Ser Ile Thr Leu TyrSer Met Leu Leu Gln Leu Asn Phe Glu Met65                  70                  75                  80 Ala AlaMet Arg Gly Cys Trp Leu Ala Leu Asp Glu Leu His Asn Val                85                  90                  95 Arg Leu CysPhe Gln Gln Ser Leu Glu His Leu Asp Glu Ala Ser Phe            100                 105                 110 Ser Asp Ile ValSer Gly Phe Ile Glu His Ala Ala Glu Val Arg Glu        115                 120                 125 Tyr Ile Ala Gln LeuAsp Glu Ser Ser Ala Ala     130                 135This protein or polypeptide is about 16 kDa and has a pI of 4.45.

Fragments of the above hypersensitive response elicitor polypeptide orprotein are encompassed by the present invention.

Suitable fragments can be produced by several means. In the first,subclones of the gene encoding the elicitor protein of the presentinvention are produced by conventional molecular genetic manipulation bysubcloning gene fragments. The subclones then are expressed in vitro orin vivo in bacterial cells to yield a smaller protein or peptide thatcan be tested for elicitor activity according to the procedure describedbelow.

As an alternative, fragments of an elicitor protein can be produced bydigestion of a full-length elicitor protein with proteolytic enzymeslike chymotrypsin or Staphylococcus proteinase A, or trypsin. Differentproteolytic enzymes are likely to cleave elicitor proteins at differentsites based on the amino acid sequence of the elicitor protein. Some ofthe fragments that result from proteolysis may be active elicitors ofresistance.

In another approach, based on knowledge of the primary structure of theprotein, fragments of the elicitor protein gene may be synthesized byusing the PCR technique together with specific sets of primers chosen torepresent particular portions of the protein. These then would be clonedinto an appropriate vector for increased expression of a truncatedpeptide or protein.

Chemical synthesis can also be used to make suitable fragments. Such asynthesis is carried out using known amino acid sequences for theelicitor being produced. Alternatively, subjecting a full lengthelicitor to high temperatures and pressures will produce fragments.These fragments can then be separated by conventional procedures (e.g.,chromatography, SDS-PAGE).

Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theproperties, secondary structure and hydropathic nature of thepolypeptide. For example, a polypeptide may be conjugated to a signal(or leader) sequence at the N-terminal end of the protein whichco-translationally or post-translationally directs transfer of theprotein. The polypeptide may also be conjugated to a linker or othersequence for ease of synthesis, purification, or identification of thepolypeptide.

Suitable DNA molecules are those that hybridize to a DNA moleculecomprising a nucleotide sequence of SEQ. ID. Nos. 1 and 3, understringent conditions. An example of suitable high stringency conditionsis when hybridization is carried out at 65° C. for 20 hours in a mediumcontaining 1 M NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.1% sodiumdodecyl sulfate, 0.2% ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovineserum albumin, 50 μm g/ml E. coli DNA. However, any DNA moleculeshybridizing to a DNA molecule comprising the nucleotide sequences ofSEQ. ID. Nos. 1 and 3, under such stringent conditions must not beidentical to the nucleic acids encoding the hypersensitive responseelicitor proteins or polypeptides of E. amylovora (as disclosed by Wei,Z.-M., et al, “Harpin, Elicitor of the Hypersensitive Response Producedby the Plant Pathogen Erwinia amylovora,” Science 257:85-88 (1992),which is hereby incorporated by reference), Erwinia chrysanthemi (asdisclosed by Bauer, et. al., “Erwinia chrysanthemi Harpin_(Ech):Soft-Rot Pathogenesis,” MPMI 8(4): 484-91 (1995), which is herebyincorporated by reference), Erwinia carotovora (as disclosed by Cui, et.al., “The RsmA⁻ Mutants of Erwinia carotovora subsp. carotovora StrainEcc71 Overexpress hrpN_(Ecc) and Elicit a Hypersensitive Reaction-likeResponse in Tobacco Leaves,” MPMI 9(7): 565-73 (1966), which is herebyincorporated by reference), Erwinia stewartii (as disclosed by Ahmad,et. al., “Harpin is not Necessary for the Pathogenicity of Erwiniastewartii on Maize,” 8th Int'l. Cong. Molec. Plant-Microb. Inter. Jul.14-19, 1996 and Ahmad, et. al., “Harpin is not Necessary for thePathogenicity of Erwinia stewartii on Maize,” Ann. Mtg. Am. Phytopath.Soc. Jul. 27-31, 1996), which are hereby incorporated by reference), andPseudomonas syringae pv. syringae (WO 94/26782 to Cornell ResearchFoundation, Inc., which is hereby incorporated by reference).

The protein or polypeptide of the present invention is preferablyproduced in purified form (preferably at least about 80%, morepreferably 90%, pure) by conventional techniques. Typically, the proteinor polypeptide of the present invention is secreted into the growthmedium of recombinant host cells. Alternatively, the protein orpolypeptide of the present invention is produced but not secreted intogrowth medium. In such cases, to isolate the protein, the host cell(e.g., E. coli) carrying a recombinant plasmid is propagated, lysed bysonication, heat, or chemical treatment, and the homogenate iscentrifuged to remove bacterial debris. The supernatant is thensubjected to sequential ammonium sulfate precipitation. The fractioncontaining the polypeptide or protein of the present invention issubjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the proteins. If necessary, theprotein fraction may be further purified by HPLC.

The DNA molecule encoding the hypersensitive response elicitorpolypeptide or protein can be incorporated in cells using conventionalrecombinant DNA technology. Generally, this involves inserting the DNAmolecule into an expression system to which the DNA molecule isheterologous (i.e. not normally present). The heterologous DNA moleculeis inserted into the expression system or vector in proper senseorientation and correct reading frame. The vector contains the necessaryelements for the transcription and translation of the insertedprotein-coding sequences.

U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporatedby reference, describes the production of expression systems in the formof recombinant plasmids using restriction enzyme cleavage and ligationwith DNA ligase. These recombinant plasmids are then introduced by meansof transformation and replicated in unicellular cultures includingprocaryotic organisms and eucaryotic cells grown in tissue culture.

Recombinant genes may also be introduced into viruses, such as vaccinavirus. Recombinant viruses can be generated by transfection of plasmidsinto cells infected with virus.

Suitable vectors include, but are not limited to, the following viralvectors such as lambda vector system gt11, gt WES.tB, Charon 4, andplasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9,pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/−or KS +/− (see “Stratagene Cloning Systems” Catalog (1993) fromStratagene, La Jolla, Calif., which is hereby incorporated byreference), pQE, pIH821, pGEX, pET series (see F. W. Studier et. al.,“Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” GeneExpression Technology vol. 185 (1990), which is hereby incorporated byreference), and any derivatives thereof. Recombinant molecules can beintroduced into cells via transformation, particularly transduction,conjugation, mobilization, or electroporation. The DNA sequences arecloned into the vector using standard cloning procedures in the art, asdescribed by Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which ishereby incorporated by reference.

A variety of host-vector systems may be utilized to express theprotein-encoding sequence(s). Primarily, the vector system must becompatible with the host cell used. Host-vector systems include but arenot limited to the following: bacteria transformed with bacteriophageDNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containingyeast vectors; mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g., baculovirus); and plant cells infected by bacteria. Theexpression elements of these vectors vary in their strength andspecificities. Depending upon the host-vector system utilized, any oneof a number of suitable transcription and translation elements can beused.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (mRNA)translation).

Transcription of DNA is dependent upon the presence of a promotor whichis a DNA sequence that directs the binding of RNA polymerase and therebypromotes mRNA synthesis. The DNA sequences of eucaryotic promotorsdiffer from those of procaryotic promotors. Furthermore, eucaryoticpromotors and accompanying genetic signals may not be recognized in ormay not function in a procaryotic system, and, further, procaryoticpromoters are not recognized and do not function in eucaryotic cells.

Similarly, translation of mRNA in procaryotes depends upon the presenceof the proper procaryotic signals which differ from those of eucaryotes.Efficient translation of mRNA in procaryotes requires a ribosome bindingsite called the Shine-Dalgarno (“SD”) sequence on the mRNA. Thissequence is a short nucleotide sequence of mRNA that is located beforethe start codon, usually AUG, which encodes the amino-terminalmethionine of the protein. The SD sequences are complementary to the3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding ofmRNA to ribosomes by duplexing with the rRNA to allow correctpositioning of the ribosome. For a review on maximizing gene expression,see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which ishereby incorporated by reference.

Promotors vary in their “strength” (i.e. their ability to promotetranscription). For the purposes of expressing a cloned gene, it isdesirable to use strong promoters in order to obtain a high level oftranscription and, hence, expression of the gene. Depending upon thehost cell system utilized, any one of a number of suitable promotors maybe used. For instance, when cloning in E coli, its bacteriophages, orplasmids, promotors such as the T7 phage promoter, lac promotor, trppromotor, recA promotor, ribosomal RNA promotor, the P_(R) and P_(L)promoters of coliphage lambda and others, including but not limited, tolacUV5, ompF, bla, lpp, and the like, may be used to direct high levelsof transcription of adjacent DNA segments. Additionally, a hybridtrp-lacUV5 (tac) promotor or other E. coli promotors produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen whichinhibit the action of the promotor unless specifically induced. Incertain operations, the addition of specific inducers is necessary forefficient transcription of the inserted DNA. For example, the lac operonis induced by the addition of lactose or IPTG(isopropylthio-beta-D-galactoside). A variety of other operons, such astrp, pro, etc., are under different controls.

Specific initiation signals are also required for efficient genetranscription and translation in procaryotic cells. These transcriptionand translation initiation signals may vary in “strength” as measured bythe quantity of gene specific messenger RNA and protein synthesized,respectively. The DNA expression vector, which contains a promotor, mayalso contain any combination of various “strong” transcription and/ortranslation initiation signals. For instance, efficient translation inE. coli requires an SD sequence about 7-9 bases 5′ to the initiationcodon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATGcombination that can be utilized by host cell ribosomes may be employed.Such combinations include but are not limited to the SD-ATG combinationfrom the cro gene or the N gene of coliphage lambda, or from the E. colitryptophan E, D, C, B or A genes. Additionally, any SD-ATG combinationproduced by recombinant DNA or other techniques involving incorporationof synthetic nucleotides may be used.

Once the isolated DNA molecule encoding the hypersensitive responseelicitor polypeptide or protein has been cloned into an expressionsystem, it is ready to be incorporated into a host cell. Suchincorporation can be carried out by the various forms of transformationnoted above, depending upon the vector/host cell system. Suitable hostcells include, but are not limited to, bacteria, virus, yeast, mammaliancells, insect, plant, and the like.

The present invention further relates to methods of imparting diseaseresistance to plants, enhancing plant growth, and/or effecting insectcontrol for plants. These methods involve applying a hypersensitiveresponse elicitor polypeptide or protein in a non-infectious form to allor part of a plant or a plant seed under conditions where thepolypeptide or protein contacts all or part of the cells of the plant orplant seed. Alternatively, the hypersensitive response elicitor proteinor polypeptide can be applied to plants such that seeds recovered fromsuch plants themselves are able to impart disease resistance in plants,to enhance plant growth, and/or to effect insect control.

As an alternative to applying a hypersensitive response elicitorpolypeptide or protein to plants or plant seeds in order to impartdisease resistance in plants, to effect plant growth, and/or to controlinsects on the plants or plants grown from the seeds, transgenic plantsor plant seeds can be utilized. When utilizing transgenic plants, thisinvolves providing a transgenic plant transformed with a DNA moleculeencoding a hypersensitive response elicitor polypeptide or protein andgrowing the plant under conditions effective to permit that DNA moleculeto impart disease resistance to plants, to enhance plant growth, and/orto control insects. Alternatively, a transgenic plant seed transformedwith a DNA molecule encoding a hypersensitive response elicitorpolypeptide or protein can be provided and planted in soil. A plant isthen propagated from the planted seed under conditions effective topermit that DNA molecule to impart disease resistance to plants, toenhance plant growth, and/or to control insects.

The embodiment of the present invention where the hypersensitiveresponse elicitor polypeptide or protein is applied to the plant orplant seed can be carried out in a number of ways, including: 1)application of an isolated elicitor polypeptide or protein; 2)application of bacteria which do not cause disease and are transformedwith genes encoding a hypersensitive response elicitor polypeptide orprotein; and 3) application of bacteria which cause disease in someplant species (but not in those to which they are applied) and naturallycontain a gene encoding the hypersensitive response elicitor polypeptideor protein.

In one embodiment of the present invention, the hypersensitive responseelicitor polypcptide or protein of the present invention can be isolatedfrom Erwinia amylovora as described in the Examples infra. Preferably,however, the isolated hypersensitive response elicitor polypeptide orprotein of the present invention is produced recombinantly and purifiedas described supra.

In other embodiments of the present invention, the hypersensitiveresponse elicitor polypeptide or protein of the present invention can beapplied to plants or plant seeds by applying bacteria containing genesencoding the hypersensitive response elicitor polypeptide or protein.Such bacteria must be capable of secreting or exporting the polypeptideor protein so that the elicitor can contact plant or plant seed cells.In these embodiments, the hypersensitive response elicitor polypeptideor protein is produced by the bacteria in planta or on seeds or justprior to introduction of the bacteria to the plants or plant seeds.

In one embodiment of the bacterial application mode of the presentinvention, the bacteria do not cause the disease and have beentransformed (e.g., recombinantly) with genes encoding a hypersensitiveresponse elicitor polypeptide or protein. For example, E. coli, whichdoes not elicit a hypersensitive response in plants, can be transformedwith genes encoding a hypersensitive response elicitor polypeptide orprotein and then applied to plants. Bacterial species other than E. colican also be used in this embodiment of the present invention.

In another embodiment of the bacterial application mode of the presentinvention, the bacteria do cause disease and naturally contain a geneencoding a hypersensitive response elicitor polypeptide or protein.Examples of such bacteria are noted above. However, in this embodiment,these bacteria are applied to plants or their seeds which are notsusceptible to the disease carried by the bacteria. For example, Erwiniaamylovora causes disease in apple or pear but not in tomato. However,such bacteria will elicit a hypersensitive response in tomato.Accordingly, in accordance with this embodiment of the presentinvention, Erwinia amylovora can be applied to tomato plants or seeds toenhance growth without causing disease in that species.

The method of the present invention can be utilized to treat a widevariety of plants or their seeds to impart disease resistance, enhancegrowth, and/or control insects. Suitable plants include dicots andmonocots. More particularly, useful crop plants can include: alfalfa,rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweetpotato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout,beet, parsnip, turnip, cauliflower, broccoli, turnip, radish, spinach,onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin,zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape,raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane.Examples of suitable ornamental plants are: Arabidopsis thaliana,Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemun, carnation,and zinnia.

With regard to the use of the hypersensitive response elicitor proteinor polypeptide of the present invention in imparting disease resistance,absolute immunity against infection may not be conferred, but theseverity of the disease is reduced and symptom development is delayed.Lesion number, lesion size, and extent of sporulation of fungalpathogens are all decreased. This method of imparting disease resistancehas the potential for treating previously untreatable diseases, treatingdiseases systemically which might not be treated separately due to cost,and avoiding the use of infectious agents or environmentally harmfulmaterials.

The method of imparting pathogen resistance to plants in accordance withthe present invention is useful in imparting resistance to a widevariety of pathogens including viruses, bacteria, and fungi. Resistance,inter alia, to the following viruses can be achieved by the method ofthe present invention: Tobacco mosaic virus and Tomato mosaic virus.Resistance, inter alia, to the following bacteria can also be impartedto plants in accordance with present invention: Pseudomonas solancearum,Pseudomonas syringae pv. tabaci, and Xanthamonas campestris pv.pelargonii. Plants can be made resistant, inter alia, to the followingfungi by use of the method of the present invention: Fusarium oxysporumand Phytophthora infestans.

With regard to the use of the hypersensitive response elicitor proteinor polypeptide of the present invention to enhance plant growth, variousforms of plant growth enhancement or promotion can be achieved. This canoccur as early as when plant growth begins from seeds or later in thelife of a plant. For example, plant growth according to the presentinvention encompasses greater yield, increased quantity of seedsproduced, increased percentage of seeds germinated, increased plantsize, greater biomass, more and bigger fruit, earlier fruit coloration,and earlier fruit and plant maturation. As a result, the presentinvention provides significant economic benefit to growers. For example,early germination and early maturation permit crops to be grown in areaswhere short growing seasons would otherwise preclude their growth inthat locale. Increased percentage of seed germination results inimproved crop stands and more efficient seed use. Greater yield,increased size, and enhanced biomass production allow greater revenuegeneration from a given plot of land.

Another aspect of the present invention is directed to effecting anyform of insect control for plants. For example, insect control accordingto the present invention encompasses preventing insects from contactingplants to which the hypersensitive response elicitor has been applied,preventing direct insect damage to plants by feeding injury, causinginsects to depart from such plants, killing insects proximate to suchplants, interfering with insect larval feeding on such plants,preventing insects from colonizing host plants, preventing colonizinginsects from releasing phytotoxins, etc. The present invention alsoprevents subsequent disease damage to plants resulting from insectinfection.

The present invention is effective against a wide variety of insects.European corn borer is a major pest of corn (dent and sweet corn) butalso feeds on over 200 plant species including green, wax, and limabeans and edible soybeans, peppers, potato, and tomato plus many weedspecies. Additional insect larval feeding pests which damage a widevariety of vegetable crops include the following: beet armyworm, cabbagelooper, corn ear worm, fall armyworm, diamondback moth, cabbage rootmaggot, onion maggot, seed corn maggot, pickleworm (melonworm), peppermaggot, and tomato pinworm. Collectively, this group of insect pestsrepresents the most economically important group of pests for vegetableproduction worldwide.

The method of the present invention involving application of thehypersensitive response elicitor polypeptide or protein can be carriedout through a variety of procedures when all or part of the plant istreated, including leaves, stems, roots, propagules (e.g., cuttings),etc. This may (but need not) involve infiltration of the hypersensitiveresponse elicitor polypeptide or protein into the plant. Suitableapplication methods include high or low pressure spraying, injection,and leaf abrasion proximate to when elicitor application takes place.When treating plant seeds, in accordance with the application embodimentof the present invention, the hypersensitive response elicitor proteinor polypeptide can be applied by low or high pressure spraying, coating,immersion, or injection. Other suitable application procedures can beenvisioned by those skilled in the art provided they are able to effectcontact of the hypersensitive response elicitor polypeptide or proteinwith cells of the plant or plant seed. Once treated with thehypersensitive response elicitor of the present invention, the seeds canbe planted in natural or artificial soil and cultivated usingconventional procedures to produce plants. After plants have beenpropagated from seeds treated in accordance with the present invention,the plants may be treated with one or more applications of thehypersensitive response elicitor protein or polypeptide to impartdisease resistance to plants, to enhance plant growth, and/or to controlinsects on the plants.

The hypersensitive response elicitor polypeptide or protein can beapplied to plants or plant seeds in accordance with the presentinvention alone or in a mixture with other materials. Alternatively, thehypersensitive response elicitor polypeptide or protein can be appliedseparately to plants with other materials being applied at differenttimes.

A composition suitable for treating plants or plant seeds in accordancewith the application embodiment of the present invention contains ahypersensitive response elicitor polypeptide or protein in a carrier.Suitable carriers include water, aqueous solutions, slurries, or drypowders. In this embodiment, the composition contains greater than 500nM hypersensitive response elicitor polypeptide or protein.

Although not required, this composition may contain additional additivesincluding fertilizer, insecticide, fungicide, nematacide, and mixturesthereof. Suitable fertilizers include (NH₄)₂NO₃. An example of asuitable insecticide is Malathion. Useful fungicides include Captan.

Other suitable additives include buffering agents, wetting agents,coating agents, and abrading agents. These materials can be used tofacilitate the process of the present invention. In addition, thehypersensitive response elicitor polypeptide or protein can be appliedto plant seeds with other conventional seed formulation and treatmentmaterials, including clays and polysaccharides.

In the alternative embodiment of the present invention involving the useof transgenic plants and transgenic seeds, a hypersensitive responseelicitor polypeptide or protein need not be applied topically to theplants or seeds. Instead, transgenic plants transformed with a DNAmolecule encoding a hypersensitive response elicitor polypeptide orprotein are produced according to procedures well known in the art

The vector described above can be microinjected directly into plantcells by use of micropipettes to transfer mechanically the recombinantDNA. Crossway, Mol. Gen. Genetics, 202:179-85 (1985), which is herebyincorporated by reference. The genetic material may also be transferredinto the plant cell using polyethylene glycol. Krens, et al., Nature,296:72-74 (1982), which is hereby incorporated by reference.

Another approach to transforming plant cells with a gene which impartsresistance to pathogens is particle bombardment (also known as biolistictransformation) of the host cell. This can be accomplished in one ofseveral ways. The first involves propelling inert or biologically activeparticles at cells. This technique is disclosed in U.S. Pat. Nos.4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which arehereby incorporated by reference. Generally, this procedure involvespropelling inert or biologically active particles at the cells underconditions effective to penetrate the outer surface of the cell and tobe incorporated within the interior thereof. When inert particles areutilized, the vector can be introduced into the cell by coating theparticles with the vector containing the heterologous DNA.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried bacterial cells containingthe vector and heterologous DNA) can also be propelled into plant cells.

Yet another method of introduction is fusion of protoplasts with otherentities, either minicells, cells, lysosomes or other fusiblelipid-surfaced bodies. Fraley, et al., Proc. Natl. Acad. Sci. USA,79:1859-63 (1982), which is hereby incorporated by reference.

The DNA molecule may also be introduced into the plant cells byelectroporation. Fromm et al., Proc. Natl. Acad. Sci. USA, 82:5824(1985), which is hereby incorporated by reference. In this technique,plant protoplasts are electroporated in the presence of plasmidscontaining the expression cassette. Electrical impulses of high fieldstrength reversibly permeabilize biomembranes allowing the introductionof the plasmids. Electroporated plant protoplasts reform the cell wall,divide, and regenerate.

Another method of introducing the DNA molecule into plant cells is toinfect a plant cell with Agrobacterium tumefaciens or A. rhizogenespreviously transformed with the gene. Under appropriate conditions knownin the art, the transformed plant cells are grown to form shoots orroots, and develop further into plants. Generally, this procedureinvolves inoculating the plant tissue with a suspension of bacteria andincubating the tissue for 48 to 72 hours on regeneration medium withoutantibiotics at 25-28° C.

Agrobacterium is a representative genus of the gram-negative familyRhizobiaceae. Its species are responsible for crown gall (A.tumefaciens) and hairy root disease (A. rhizogenes). The plant cells incrown gall tumors and hairy roots are induced to produce amino acidderivatives known as opines, which are catabolized only by the bacteria.The bacterial genes responsible for expression of opines are aconvenient source of control elements for chimeric expression cassettes.In addition, assaying for the presence of opines can be used to identifytransformed tissue.

Heterologous genetic sequences can be introduced into appropriate plantcells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid ofA. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells oninfection by Agrobacterium and is stably integrated into the plantgenome. J. Schell, Science, 237:1176-83 (1987), which is herebyincorporated by reference.

After transformation, the transformed plant cells must be regenerated.

Plant regeneration from cultured protoplasts is described in Evans etal., Handbook of Plant Cell Cultures. Vol. 1: (MacMillan Publishing Co.,New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. 1, 1984, and Vol. III(1986), which are hereby incorporated by reference.

It is known that practically all plants can be regenerated from culturedcells or tissues, including but not limited to, all major species ofsugarcane, sugar beets, cotton, fruit trees, and legumes.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts or a petri platecontaining transformed explants is first provided. Callus tissue isformed and shoots may be induced from callus and subsequently rooted.Alternatively, embryo formation can be induced in the callus tissue.These embryos germinate as natural embryos to form plants. The culturemedia will generally contain various amino acids and hormones, such asauxin and cytokinins. It is also advantageous to add glutamic acid andproline to the medium, especially for such species as corn and alfalfa.Efficient regeneration will depend on the medium, on the genotype, andon the history of the culture. If these three variables are controlled,then regeneration is usually reproducible and repeatable.

After the expression cassette is stably incorporated in transgenicplants, it can be transferred to other plants by sexual crossing. Any ofa number of standard breeding techniques can be used, depending upon thespecies to be crossed.

Once transgenic plants of this type are produced, the plants themselvescan be cultivated in accordance with conventional procedure with thepresence of the gene encoding the hypersensitive response elicitorresulting in disease resistance, enhanced plant growth, and/or controlof insects on the plant. Alternatively, transgenic seeds are recoveredfrom the transgenic plants. These seeds can then be planted in the soiland cultivated using conventional procedures to produce transgenicplants. The transgenic plants are propagated from the planted transgenicseeds under conditions effective to impart disease resistance to plants,to enhance plant growth, and/or to control insects. While not wishing tobe bound by theory, such disease resistance, growth enhancement, and/orinsect control may be RNA mediated or may result from expression of theelicitor polypeptide or protein.

When transgenic plants and plant seeds are used in accordance with thepresent invention, they additionally can be treated with the samematerials as are used to treat the plants and seeds to which ahypersensitive response elicitor polypeptide or protein is applied.These other materials, including hypersensitive response elicitors, canbe applied to the transgenic plants and plant seeds by the above-notedprocedures, including high or low pressure spraying, injection, coating,and immersion. Similarly, after plants have been propagated from thetransgenic plant seeds, the plants may be treated with one or moreapplications of the hypersensitive response elicitor to impart diseaseresistance, enhance growth, and/or control insects. Such plants may alsobe treated with conventional plant treatment agents (e.g., insecticides,fertilizers, etc.).

Another aspect of the present invention is to utilize the subjectelicitor proteins or polypeptides to design molecules that willinactivate, destroy, or bind to these proteins or polypeptides. Sincethese elicitors are found in plant pathogens, particularly Erwiniaamylovora, the pathogens themselves can be neutralized by the designedmolecules so that disease and/or hypersensitive response is prevented oraltered. Examples of disease preventing molecules are antibodies, suchas monoclonal or polyclonal antibodies, raised against the elicitorproteins or polypeptides of the present invention or binding portionsthereof. Other examples of disease preventing molecules include antibodyfragments, half-antibodies, hybrid derivatives, probes, and othermolecular constructs.

Monoclonal antibody production may be effected by techniques which arewell-known in the art. Basically, the process involves first obtainingimmune cells (lymphocytes) from the spleen of a mammal (e.g., mouse)which has been previously immunized, either in vivo or in vitro, withthe antigen of interest (e.g., an elicitor protein or polypeptide of thepresent invention or binding portions thereof). The antibody-secretinglymphocytes are then fused with (mouse) myeloma cells or transformedcells, which are capable of replicating indefinitely in cell culture,thereby producing an immortal, immunoglobulin-secreting cell line. Theresulting fused cells, or hybridomas, are cultured, and the resultingcolonies screened for the production of the desired monoclonalantibodies. Colonies producing such antibodies are cloned, and growneither in vivo or in vitro to produce large quantities of antibody. Adescription of the theoretical basis and practical methodology of fusingsuch cells is set forth in Kohler and Milstein, Nature 256:495 (1975),which is hereby incorporated by reference.

Mammalian lymphocytes are immunized by in vivo immunization of theanimal (e.g., a mouse) with the elicitor proteins or polypeptides of thepresent invention or binding portions thereof. Such immunizations arerepeated as necessary at intervals of up to several weeks to obtain asufficient titer of antibodies. Following the last antigen boost, theanimals are sacrificed and spleen cells removed.

Fusion with mammalian mycloma cells or other fusion partners capable ofreplicating indefinitely in cell culture is effected by standard andwell-known techniques, for example, by using polyethylene glycol (“PEG”)or other fusing agents (See Milstein and Kohler, Eur. J. Immunol. 6:511(1976), which is hereby incorporated by reference). This immortal cellline, which is preferably murine, but may also be derived from cells ofother mammalian species, including but not limited to rats, is selectedto be deficient in enzymes necessary for the utilization of certainnutrients, to be capable of rapid growth, and to have good fusioncapability. Many such cell lines are known to those skilled in the art,and others are regularly described.

Procedures for raising polyclonal antibodies are also well known.Typically, such antibodies can be raised by administering the elicitorproteins or polypeptides of the present invention or binding portionsthereof subcutaneously to New Zealand white rabbits which have firstbeen bled to obtain pre-immune serum. The antigens can be injected at atotal volume of 100 μl per site at six different sites. Each injectedmaterial will contain synthetic surfactant adjuvant pluronic polyols, orpulverized acrylamide gel containing the protein or polypeptide afterSDS-polyacrylamide gel electrophoresis. The rabbits are then bled twoweeks after the first injection and periodically boosted with the sameantigen three times every six weeks. A sample of serum is then collected10 days after each boost. Polyclonal antibodies are then recovered fromthe serum by affinity chromatography using the corresponding antigen tocapture the antibody. Ultimately, the rabbits are euthenized withpentobarbital 150 mg/Kg IV. This and other procedures for raisingpolyclonal antibodies are disclosed in E. Harlow, et. al., editors,Antibodies: A Laboratory Manual (1988), which is hereby incorporated byreference.

In addition to utilizing whole antibodies, the processes of the presentinvention encompass use of binding portions of such antibodies. Suchbinding portions include Fab fragments, F(ab′)₂ fragments, and Fvfragments. These antibody fragments can be made by conventionalprocedures, such as proteolytic fragmentation procedures, as describedin J. Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-118(N.Y. Academic Press 1983), which is hereby incorporated by reference.

Alternatively, the processes of the present invention can utilize probesor ligands found either in nature or prepared synthetically byrecombinant DNA procedures or other biological or molecular procedures.Suitable probes or ligands are molecules which bind to the elicitorproteins or polypeptides of the present invention or binding portionsthereof.

A virulence (avr) genes (see Vivian, A., et al, Microbiology,143:693-704 (1997); Leach, J. E., et al., Annu. Rev. Phytopathol.,34:153-179 (1996); Dangl, J. L. “Bacterial Pathogenesis of Plants andAnimals: Molecular and Cellular Mechanisms,” in Current Topics inMicrobiology and Immunology, Dangl. J. L., ed. (Springer, Berlin), Vol.192, pp. 99-118 (1994), which are hereby incorporated by reference)generate signals that trigger defense responses leading to diseaseresistance in plants with corresponding resistance (R) genes. Typically,avr genes are isolated by expressing a cosmid library from one pathogenin another pathogen and screening for narrowed host range. avr genestraditionally have been considered as negative determinants of hostspecificity at the race-cultivar level, but some, including the avrElocus from the bacterial speck pathogen Pseudomonas syringae pathovar(pv.) tomato (Kobayashi, D. Y., et al., Proc. Natl. Acad. Sci. USA,86:157-61 (1989), which is hereby incorporated by reference), restricthost range at the pathovar-species or species—species level (Whalen, M.C., et al., Proc. Natl. Acad. Sci. USA, 85:6743-47 (1988); Swarup, S.,et al., Mol. Plant-Microbe Interact., 5:204-13 (1992), which are herebyincorporated by reference). Many avr genes, including avrE, are Hrpregulated. avrE and avrPphE (Mansfield, J., et al., Mol. Plant-MicrobeInteract., 7:726-39 (1994), which is hereby incorporated by reference)are physically linked to hrp genes.

When expressed in trans, the avrE locus renderes P. syringae pv.glycinea, which causes bacterial blight of soybean, a virulent in allcultivars (Lorang, J. M., et al., Mol. Plant-Microbe Interact., 8:49-57(1995), which is hereby incorporated by reference). The locus comprisestwo convergent transcription units, one preceded by a putative σ⁵⁴promoter and the other by a hrp box, a sequence found upstream of manyhrp and avr genes that are positively regulated by the alternate sigmafactor HrpL (Innes, R. W., et al., J. Bacteriol., 175:4859-69 (1993);Shen, H., et al., J. Bacterol., 175:5916-24 (1993); Xiao, Y., et al., J.Bacteriol., 176:3089-91 (1994), which are hereby incorporated byreference). Expression of both transcripts require HrpL. The avrE locuscontributes quantitatively to the virulence in tomato leaves of P.syringae pv. tomato strain PT23, but not of strain DC3000 (Lorang, J.M., et al., Mol. Plant-Microbe Interact., 8:49-57 (1995); Lorang, J. M.,et al., Mol. Plant-Microbe Interact. 7:508-515 (1994)).

Thus, avr genes in plant pathogens bind to disease resistance genes inplants which are not susceptible to that pathogen. In view of thehomology of the DNA molecules of the present invention to avr genes inplant pathogens, these DNA molecules can be used to identifycorresponding plant disease resistance genes. Such identification iscarried out by traditional plant breeding techniques in which a pathogencarrying the avr gene is inoculated to plants in screening to trackinheritance or identify disruption of the resistance. Once identified,the resistance gene can be isolated by either of two approaches thathave proved successful in recent years (see Staskawicz et al., Science,68:661-67 (1995)). These are positional or map-based cloning andinsertional mutagenesis or transposon tagging. Because there may be noDspE-insensitive cultivars (susceptible to Pseudomonas harboring dspE;each of four soybean cultivars tested responded to dspE), map-basedcloning (which requires crosses between susceptible and resistant linesto identify the position of the resistance gene relative to other genes)may not be feasible. The preferred approach would more likely involveinsertional mutagenesis, using the dspE gene or protein in screens toidentify lines which had lost the the product of dspE due to transposontagging of the corresponding resistance gene.

EXAMPLES Example 1 Recombinant DNA Techniques

Isolation of DNA, restriction enzyme digests, ligation, transformationof Escherichia coli, and construction of and colony hybridization toscreen a P. syringae pv. tomato DC3000 genomic library were performed asdescribed by Sambrook, et al. (Sambrook, J., et al., Molecular cloning:A Laboratory manual, (Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.) (1989), which is hereby incorporated by reference). The librarywas constructed using pCPP47 (Bauer, D. W., et al., Mol. Plant-MicrobeInteract., 10:369-379 (1997), which is hereby incorporated byreference). Except where noted, E. coli DH5 and E. coli DH5α were usedas hosts for DNA clones, and pBluescript or pBC plasmids (Stratagene, LaJolla, Calif.) were used as vectors. E. amylovora was transformed byelectroporation as described (Bauer, D. W. in “Molecular Genetics ofPathogenicity of Erwinia amylovora: Techniques, Tools and TheirApplications”, (Ph. D. Thesis), Cornell University, Ithaca, N.Y. (1990),which is hereby incorporated by reference). Plasmids were mobilized intoE. amylovora and P. syringae using pRK2013 (Figurski, D., et al., Proc.Natl. Acad. Sci. USA 76:1648-1652 (1979), which is hereby incorporatedby reference).

Example 2 Nucleotide Sequencing and Analysis

The nucleotide sequence of the dsp region of E. amylovora strain Ea321was determined using sublcones of pCPP430 (Beer, S. V., et al., inAdvances in Molecular Genetics of Plant-Microbe Interactions, Hennecke,H., et al., eds. (Kluwer Academic Publishers, Dordrecht, TheNetherlands), pp. 53-60 (1991), which is hereby incorporated byreference). The nucleotide sequence of the avrE locus was determinedusing subelones of pCPP2357, a clone selected from a P. syringae pv.tomato DC3000 genomic cosmid library based on hybridization with thehrpRS operon of P. syringae pv. syringae, and the finding, based onpartial sequencing, that it contained the avrElocus. Nucleotidesequencing was performed by the Cornell Biotechnology SequencingFacility on a Model 377 Sequencer (Perkin Elmer/Applied BiosystemsDivision, Foster City, Calif.). Sequence assembly, analysis, andcomparisons were performed using the programs of the GCG softwarepackage, version 7.1 (Genetics Computer Groups, Inc., Madison, Wis.) andDNASTAR (DNASTAR, Inc., Madison, Wis.). Database searches were performedusing BLAST (Altschul, S. F., et al., Proc. Nat. Acad. Sci. USA,87:5509-5513 (1990) which is hereby incorporated by reference).

Example 3 Expression of DspE and DspE' in E. coli

The dspE operon was cloned in two pieces into pCPP50, a derivative ofPINIII¹¹³-A2 (Duffaud, G. D., et al. in Methods in Enzymology, Wu, R.,et al., eds. (Academic Press, New York), 153:492-50 (1987), which ishereby incorporated by reference) with an expanded polylinker, yieldingpCPP1259. Expression in pCPP1259 is driven by the Ipp promoter of E.coli, under the control of the lac operator. An intermediate clone,pCPP1244, extending from the start of the operon to the BamHI site inthe middle of dspE, also was isolated. E. coli DH5α strains containingpCPP1259 and pCPP1244 were grown in LB at 37° C. to an OD₆₂₀ of 0.3.Isopropylthio-β-D-galactoside then was added to 1 mM, and the cellsfurther incubated until reaching an OD₆₂₀ of 0.5. Cells wereconcentrated two-fold, lysed and subjected to SDS-PAGE as previouslydescribed (Sambrook, J., et al., Molecular cloning: A Laboratory Manual(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) (1989), whichis hereby incorporated by reference), using 7.5% acrylamide. Cellscontaining pCPP50 were included for comparison. Proteins were visualizedby Coomassie staining.

Example 4 Deletion Mutagenesis of dspE

1554 bp were deleted from the 5′ HindIII-BamHI fragment of dspE inpCPP1237 using unique Stul and Smal sites. The mutagenized clone thenwas inserted into the suicide vector pKNG101 (Kaniga, K., et al., Gene,109:137-42 (1991), which is hereby incorporated by reference) using E.coli SM10λpir as a host, yielding pCPP1241. The mutation, designatedΔ1554; then was transferred into E. amylovora strains using markereviction as described previously (Bogdanove, A. J., et al., J.Bacteriol., 178:1720-30 (1996), which is hereby incorporated byreference). 1521 bp were deleted from the 3′ HindIII fragment of dspE inpCPP 1246 using two BstEll sites blunted with Klenow fragment. Thismutation, Δ1521, was transferred into E. amylovora strains as above.

Example 5 Pathogenicity Assays

For E. amylovora strains, cell suspensions of 5×10⁸ colony-forming units(cfu) per ml were pipetted into wells cut in immature Bartlett pearfruit, or stabbed into Jonamac apple and cotoneaster shoots, and assayscarried out as described previously (Beer, S. V., in Methods inPhytobacteriology, Klement, Z., et al., eds. (Adadémiai Kiadoó,Budapest), pp. 373-374 (the “1990); Aldwinckle, H. S., et al.,Phytopathology, 66:1439-44 (1976), which are hereby incorporated byreference). For P. syringage pv. glycinea, panels of primary leaves of2-week-old soybean seedlings (Glycine max, cultivar Norchief) wereinfiltrated with bacterial suspensions of 8×10⁵ cfu/ml as for the HRassay, below. Plants were then covered with clear plastic bags andincubated under fluorescent lights (16 hr/day) at 22° C. for 5-7 days.Leaves were scored for necrosis and chlorosis.

Example 6 HR Assays

Tobacco leaf panels (Nicotiana tabacum L. ‘xanthi’) were infiltratedwith bacterial cell suspensions as described previously (Wei, Z. M., etal., Science, 257:85-88 (1992); Bauer, D. W., et al., Mol. Plant-MicrobeInteract., 4:493-99 (1991), which are hereby incorporated by reference).Primary leaves of 2-week-old soybean seedlings (secondary leavesemerging) were infiltrated with bacterial cell suspensions as fortobacco. Plants were scored for HR (tissue collapse) after 24-48 hr onthe laboratory bench. E. amylovora strains were suspended in 5 mM KPO₄buffer, pH 6.8, and P. syringae strains in 10 mM MgCl₂.

Example 7 GUS Assays

Cells were 1.) grown in LB to an OD₆₂₀ of 0.9-1.0; 2.) grown in LB to anOD₆₂₀ of 0.5, then washed and resuspended in a hrp-gene-inducing minimalmedium (Hrp MM; Huynh, T. V., et al., Science, 345:1374-77 (1989), whichis hereby incorporated by reference) to an OD₆₂₀ of 0.2 and incubated at21° C. for 36 hr to a final OD₆₂₀ of 0.9-1.0; or 3.) grown in LB to anOD₆₂₀ of 0.5, washed and concentrated 2-fold in 5 mM KPO₄ buffer, pH6.8, and then transferred to freshly cut wells in pear halves andincubated as for the pathogenicity assay for 36 hr. Cells were assayedfor β-glucuronidase (GUS) activity essentially according to Jefferson(Jefferson, R. A., Plant Molecular Biology Reporter, 5:387-405 (1987),which is hereby incorporated by reference). For the cells in LB or HrpMM, 50 μl were mixed with 200 μl GUS extraction buffer (50 mM NaHPO₄, pH7.0, 10 mM β-mercaptoethanol, 10 mM Na₂EDTA, 0.1% sodium laurylsarcosine, 0.1% Triton X-100) containing 2 mM 4-methylumbelliferylβ-D-glucuronide as substrate and incubated at 37° C. for 100 min. Forcells in pear fruit, the tissue surrounding the well was excised using a#4 cork borer and homogenized in 5 mM KPO₄ buffer, pH 6.8. 200 μl ofhomogenate was mixed with 800 μl of GUS extraction buffer with substrateand incubated as above. Reactions were stopped by adding Na₂CO₃ to afinal concentration of 0.2 M in a total volume of 2 ml. Fluorescence wasmeasured using a TKO 100 Mini-Fluorometer (Hoefer ScientificInstruments, San Francisco, Calif.). For all samples, cell concentrationwas estimated by dilution plating, and fluorometric readings wereconverted to pmole of substrate hydrolyzed/10⁸ cfu/min, after Miller(Miller, J. H., A Short Course in Bacterial Genetics: A LaboratoryManual and Handbook for Escherichia coli and Related Bacteria (ColdSpring Harbor Laboratory Press, Plainview, N.Y.) (1992), which is herebyincorporated by reference).

Example 8 The “Disease-specific” (dsp) Region of E. amylovora Consistsof a 6.6 kb, two-gene Operon.

Mapping of previous transposon insertions (Steinberger, E. M., et al.,Mol. Plant-Microbe Interact., 1:135-44 (1988), which is herebyincorporated by reference) that abolish pathogenicity but notHR-eliciting ability confirmed the presence of the “disease specific”(dsp) region downstream of the hrpN gene in strain Ea321 as reported instrain CFBP1430 (Barny, A. M., et al., Mol. Microbiol., 4:777-86 (1990),which is hereby incorporated by referece). The sequence of approximately15 kb of DNA downstream of hrpN from Ea321 was determined, revealingseveral open reading frames (ORFs' FIG. 1). One ORF, in an apparent 6.6kb operon with a smaller ORF, spanned the area to which the dspinsertions mapped. These two ORFs were designated dspE and dspF. dspE ispreceded (beginning 70 bp upstream of the initiation codon) by thesequence GGAACCN₁₅CAACATAA (SEQ. ID. No. 5), which matches theHrpL-dependent promoter consensus sequence or “hrpbox” of E. amylovora(Kim, J. H., et al., J. Bacteriol., 179:1690-97 (1997); Kim, J. H., etal., J. Bacteriol., 179:1690-97 (1997), which are hereby incorporated byreference) and strongly resembles the hrp box of P. syringae hrp and avrgenes (Xiao, Y., et al., J. Bacteriol., 176:3089-91 (1994), which ishereby incorporated by reference). Immediately downstream of dspF isA/T-rich DNA, followed by an ORF (ORF7) highly similar to the Salmonellatyphimurium gene spvR, a member of the lysR family of regulatory genes(Caldwell, A. L. & Gulig, P. A., J. Bacteriol, 173:7176-85 (1991), whichis hereby incorporated by reference). Immediately upstream of the dspEoperon is a Hrp-regulated gene, hrpW, encoding a novel harpin.

The deduced product of dspE contains 1838 amino acid residues and ishydrophilic. The predicted molecular weight, 198 kD, was confirmed byexpression in E. coli (FIG. 2). Expression of an intermediate clonecontaining only the 5′ half of dspE yielded a protein of correspondingpredicted mobility, suggesting that the N-terminal half of the proteinmight form an independently stable domain. DspF, predicted to be 16 kD,acidic (pl, 4.45), and predominantly α-helical, with amphipathic αhelices in its C-terminus, is physically similar to virulence factorchaperones of animal-pathogenic bacteria (Wattiau, P., et al., Mol.Microbiol., 20:255-62 (1996), which is hereby incorporated byreference).

Example 9 dspE is Required for Fire Blight

Two in-frame deletions within dspE (FIG. 1) were made in Ea321 and Ea273(low- and high-virulence strains, respectively). The first (Δ1554)corresponds to amino acid residues G₂₀₃ to G₇₂₀, and the second (Δ1521)to amino acid residues T₁₀₆₄ to V₁₅₇₀. Each deletion abolished theability of both strains to cause fire blight when inoculated to inmaturepear fruit (FIG. 3), apple shoots, or cotoneaster shoots. Δ1554 wascomplemented by a clone carrying only the overlapping 5′ half of dspE,further suggesting that the N-terminus of the protein forms a stabledomain (FIGS. 1 and 3).

Example 10 The dspE Operon Contributes Quantitatively and in aStrain-dependent Fashion to HR Elicitation by E. amylovora in Tobaccoand is not Required for HR Elicitation by E. amylovora in Soybean.

Transposon insertions in the dsp region reduce the ability of E.amylovora to elicit the HR in tobacco (Barn, A. M., et al., Mol.Microbiol., 4:777-86 (1990), which is hereby incorporated by reference).Dilution series of suspensions of dspEΔ1554 mutant strains of Ea321 andEa273 were infiltrated into tobacco leaves alongside their wild-typeparents to assess the role of dspe in HR elicitation (FIG. 3). Allstrains were capable of eliciting the HR, but Ea321 dspEΔ1554, on aper-cell basis, was roughly one-tenth as effective as the wild-type ineliciting tissue collapse. There was no noticeable difference inHR-eliciting activity, however, between Ea273 and Ea273dspEΔ1554.Ea321dspEΔ1554 elicited wild-type HR in Acme, Centennial, Harasoy, andNorchief soybean leaves (FIG. 3).

Example 11 The dspE Operon is Hrp-regulated

A promoterless uidA gene construct was cloned downstream of the dspEfragment in pCPP1241 that was used to introduce the Δ1554 mutation(FIG. 1) into wild-type strains of E. amylovora (this construct consistsof a 3′-truncated dspE gene with the internal deletion). The resultingplasmid, pCPP1263, was mobilized into Ea321 and Ea273. Pathogenicstrains, in which plasmid integration had preserved an intact copy ofdspE, and non-pathogenic strains, in which the native copy of dspE hadbeen mutated, were isolated. All strains were assayed for GUS activityin Luria Bertani medium (LB) and in Hrp MM, and pathogenic strains wereassayed for activity in pear fruit. High levels of activity wereobtained from strains incubated in Hrp MM and pear, but not LB. Thelevel of expression in Hrp MM was equivalent to that of a hrcV-uidAfusion (“G73”, Wei, et al., J. Bacteriol., 177:6201-10 (1995), which ishereby incorporated by reference) used as a positive control. There wereno significant differences in levels of expression of the dspE-uidAfusion in the wild-type and dspE mutant backgrounds, indicating thatdspE likely is not autoregulated. Expression of the dspE-uidA fusion inhrpL mutants of Ea321 and Ea273 in hrp MM was two orders of magnitudelower than that in HrpL+strains. Data for Ea273 and derivatives areshown in FIG. 4.

Example 12 dspE and dspF are Homologous With Genes in the avrE Locus ofPseudomonas syringage pv. Tomato

A BLAST (Altschul, S. F., et al., J. Mol. Biol., 215:403-10 (1990),which is hereby incorporated by reference) search of the geneticdatabases revealed similarity of dspE to a partial sequence of the avrElocus of P. syringae pv. tomato (Lorang, J. M., et al., Mol.Plant-Microbe Interact, 8:49-57 (1995), which is hereby incorporated byreference). A cosmid library of P. syringae pv. tomato DC3000 genomicDNA was constructed, and a clone overlapping the hrp gene cluster andcontaining the avrE locus was isolated (pCPP2357). The completenucleotide sequence of the avrElocus was determined, revealing thehomolog of dspE (encoding a 195 kD, 1795 amino acid protein of 30%identity) alone in an operon previously designated transcription unitIII, and a homolog of dspF (encoding a 14 kD, a 129 amino acid proteinof 43% identity) at the end of the juxtaposed and opposing operonpreviously designated transcription unit IV (FIG. 1). These genes aredesignated avrE and avrF. The C-terminal half of the DspE and AvrEalignment (from V₈₄₅ of DspE) shows greater conservation (33% identity)than the N-terminal half (26% identity). AvrE contains a motif (aaresidues A₄₅₀ to T₄₅₇) conserved in ATP— or GTP-binding proteins(“P-loop”; Saraste, M., et al., Trends Biochem. Sci., 15:430-34 (1990),which is hereby incorporated by reference). This motif is not conservedin DspE, however, and its functional significance in AvrE, if any, isunclear. Amino acid identities are distributed equally throughout theDspF and AvrF alignment, and AvrF shares the predicted physicalcharacteristics of DspF. Upstream of avrF, completing the operon, is a2.5 kb gene with no similarity to sequences in the genetic databases.

Example 13 The dspE Operon Functions as an Avirulence Locus

The dspE operon was cloned into pML 122 (Labes, M., et al., Gene,89:37-46 (1990), which is hereby incorporated by reference) downstreamof the nptll promoter, and this construct, pCPP1250, was mobilized intoP. syringae pv. glycinea race 4. The resulting strain, but not a controlstrain containing pML 122, elicited the HR in soybean cultivars Acme,Centennial, Harasoy, and Norchief; in Norchief plants incubated underconducive conditions, race 4 harboring pCPP1250 failed to cause symptomsof disease, while the control strain caused necrosis and chlorosis thatspread from the point of inoculation (FIG. 5).

Example 14 avrE Complements dspE Mutations

Cosmid pCPP2357 was mobilized into Ea321 dspE mutant strains Δ1554 andΔ1521. The resulting transconjugants were pathogenic but low invirulence. Ea321dspEΔ1521 carrying pCPP2357 with a transposon insertionin the avrE gene was non-pathogenic, demonstrating that thecomplementation observed was avrE-specific (FIGS. 1 and 5). The sameresults were observed for transconjugants of the Ea273dspEΔ1521 mutant.

Over thirty bacterial avr genes have been discovered. The plethora ofavr genes is thought to result from an “evolutionary tug-of-war” (Dangl,J. L., in Bacterial Pathogenesis of Plants and Animals: Molecular andCellular Mechanisms(Current Topics in Microbiology and Immunology),Dangl. J. L., ed. (Springer, Berlin), 192:99-118 (1994), which is herebyincorporated by reference), a reiterative process of selection,counterselection due to R genes, and modification or substitution of avrgenes that was originally discerned by Flor, who hypothesized that“during their parallel evolution host and parasite developedcomplementary genic systems” (Flor, H. H., Adv. Genet., 8:29-54 (1956),which is hereby incorporated by reference). However, only a few avrgenes (including avrE in strain PT23) play detectable roles in virulenceor pathogen fitness in their native genetic background (Lorang, J. M.,et al., Mol. Plant-Microbe Interact., 7:508-15 (1994); Kearney, B., etal., Nature, 346:385-86 (1990); Swarup, S., et al., Phytopathology,81:802-808 (1991); De Feyter, R. D., et al., Mol. Plant-MicrobeInteract., 6:225-37 (1993); Ritter, C., et al., Mol. Plant-MicrobeInteract., 8:444-53 (1995), which are hereby incorporated by reference),and the selective force driving the maintenance in pathogen genomes ofmany of these host-range-limiting factors has remained a mystery. It isnow clear, though, that several Avr proteins are delivered into plantcells by the Hrp pathway (Gopalan, S., et al., Plant Celli, 8:1095-1105(1996); Tang, X., et al., Science, 274:2060-63 (1996); Scofield, S. R.,et al., Science, 274:2063-65 (1996); Leister, R. T., et al., Proc. Natl.Acad. Sci. USA, 93:15497-15502 (1996); Van Den Ackerveken, G., et al.,Cell, 87:1307-16 (1996), which are hereby incorporated by reference)and, therefore, are likely to be fundamentally virulence factors, whichinteract (directly, or indirectly through enzymatic products) with hosttargets to promote parasitism. Mutation of such targets (selectedbecause of reduced susceptibility) as well as the evolution of Rproteins that recognize the Avr proteins would force the acquisition orevolution of new or modified Avr proteins and result in theproliferation of avr genes. Cumulatively, these co-evolutionaryprocesses likely would drive a trend toward avr genes with quantitativeand redundant effects in pathogenesis rather than critically importantroles (Alfano, J. R., et al., Plant Cell, 8:1683-16988 (1996), which ishereby incorporated by reference).

It has been found that the homologs dspE and avrE contribute to diseaseto dramatically different extents. The avirulence locus can substitutetransgenerically for the pathogenicity operon, and that the avirulencefunction of dspE extends across pathogen genera as well. These findingssupport the hypothesis that avr genes have a primary function indisease. Moreover, they support and expand the coevolutionary model foravr gene proliferation discussed above, and they have practicalimplications concerning the control of fire blight and other bacterialdiseases of perennials.

One can predict from the model that the relative contribution topathogenicity of a particular factor would reflect, in part, the genetichistory of the pathogen, specifically, the degree of co-evolution withits host(s). dspE is required for pathogenicity; avrE has aquantitative, strain-dependent, virulence phenotype. Consistent with theprediction, evolution of corresponding R genes and modification oftargets of pathogen virulence factors is likely to have occurred moreoften and to a greater extent over time in the herbaceous hoststypically infects by P. syringae pathovars than in the woody hosts withwhich E. amylovora presumably evolved. Alternatively or additionally,acquisition of dspE (through evolution or horizontal transfer) by E.amylovora could have occurred relatively more recently than acquisitionof avrE by P. syringae, allowing less time for coevolution leading tomodification or the development of redundant function.

One could also hypothesize from the model that virulence factors may beconserved among pathogens, yet individually adapted to avoid detectionon a particular host. Preliminary results from Southern blothybridizations suggest that P. syringae pv. glycinea harbors an avrEhomolog, which, if functional, would support such a hypothesis.Similarly, homologs of the soybean cultivar-specific genes avrA and avrDfrom P. syringae pv. tomato exist in P. syringae pv. glycinea(Kobayashi, D. Y., et al., Proc. Natl. Acad. Sci. USA, 86:157-161(1989), which is hereby incorporated by reference).

The homology and abilities of dspE and avrE to function transgenericallyexpand the model for avr gene proliferation. Major components of anevolution toward multifactor virulence could be procurement of genesencoding novel virulence factors from heterologus pathogens, andconservation of a functionally cosmopolitan virulence factor deliverysystem (and possibly conservation of a universal Hrp-pathway-targetingsignal on the factors themselves) that would enable their deployment.Indeed, many avr genes are on plasmids and scattered in theirdistribution among pathogen strains (Dangl, J. L., in BacterialPathogenesis of Plants and Animals: Molecular and Cellular Mechanisms(Current Topics in Microbiology and Immunology), Dangl. J. L., ed.(Springer, Berlin), 192:99-118 (1994), which is hereby incorporated byreference), and individual hrp genes are conserved and eveninterchangeable (Arlat, M., et al., Mol. Plant-Microbe Interact.,4:593-601 (1991); Laby, R. J., et al., Mol. Plant-Microbe Interact.,5:412-19 (1992), which is hereby incorporated by reference). Thepresence of dspE and avrE in distinct genera suggests horizontaltransfer of an ancestral locus, and, although dspE and avrE arehomologous and hrplinked, the transgeneric function of these genessuggests that the Hrp pathways in E. amylovora and P. syringae haveremained insensitive to differences accrued in DspE and AvrE overevolution. It is predicted that even non-homologous Avr-like proteinswill function across phytopathogenic bacterial genera.

It remains to be shown whether the avirulence function of the dspE locusis Hrp-pathway-dependent. This seems likely, and it will be important todetermine the localization of the dspE and dspF gene products in theplant-bacterial interaction. The physical similarity of DspF (and AvrF)to chaperones required for type III secretion of virulence factors fromanimal-pathogenic bacteria (Wattiau, P., et al., Mol. Microbiol.,20:255-62 (1996), which is hereby incorporated by reference) isintriguing and novel in phytopathogenic bacteria. The requirement ofthese chaperones appears to be due to a role other than targeting to thesecretion pathway (Woestyn, S., et al., Mol. Microbiol., 20:1261-71(1996), which is hereby incorporated by reference): chaperones maystabilize proteins, maintain proteins in an appropriate conformation forsecretion, or prevent premature polymerization or association with otherproteins. Perhaps, DspF binds to DspE (and AvrF to AvrE) and plays asimilar role, which might be particularly important for the latterprotein due to its large size and probable multidomain nature.

The dspE operon is the first described avirulence locus in E. amylovora.A homolog of avrRxv from Xanthomonas campestris (Whalen, M. C., et al.,Proc. Natl. Acad. Sci. USA, 85:6743-47 (1988), which is herebyincorporated by reference) has been found near the dspE operon (Kim, J.F., in Molecular Characterization of a Novel Harpin and Two hrpSecretory Operons of Erwinia amylovora, and a hrp Operon of E.chrysanthemi (Ph.D. Thesis), Cornell University, Ithaca, N.Y. (1997)).Monogenic (R-gene-mediated) resistance to fire blight has not beenreported, but differential virulence of E. amyolovora strains on applecultivars has been observed (Norelli, J. L., et al., Phytopathology,74:136-39 (1984), which is hereby incorporated by reference). Also, somestrains of E. amylovora infect Rubus spp. and not pomaceous plants, andvice-versa (Starr, M. P., et al., Phytopathology, 41:915-19 (1951),which is hereby incorporated by reference). Whether the dspE operon andthe avrRxv homolog or other potential elicitors play a role in thesespecificities should be determined.

Although the dspE operon triggers defense responses in soybean whenexpressed in P. syringae pv. glycinea, it is not required for the HR ofsoybean elicited by E. amylovora. Neither is hrpN required (FIG. 3). Itis possible that E. amylovora must have one or the other, dspE or hrpN,to elicit the HR in soybean. It has been observed, however, thatpurified harpin does not elicit the HR in soybean, suggesting thealternative explanation that E. amylovora harbors another avr generecognized by this plant.

Recognition of E. amylovora avirulence signals in soybean indicates thepresence of one or more R genes that might be usefull for engineeringfire blight resistant apple and pear trees. R-gene-mediated resistanceto the apple scab pathogen Venturia inaequalis (Williams, E. B., et al.,Ann. Rev. Phytopathol., 7:223-46 (1969), which is hereby incorporated byreference) and successful transformation of apple with attacin E forcontrol of fire blight (Norelli, J. L., et al., Euphytica, 77:123-28(1994), which is hereby incorporated by reference) attest thefeasibility of such an approach. R gene-mediated resistance to applescab has been overcome in the field (Parisi, L., et al., Phytopathology,83:533-37 (1993), which is hereby incorporated by reference), but therequirement for dspE in disease favors relative durabiliity of adspE-specific R gene (Kearney, B. et al., Nature, 346:385-86 (1990),which is hereby incorporated by reference). Avirulence screening of dspEand other E. amylovora genes in pathogens of genetically tractableplants such as Arabidopsis could broaden the pool of candidate R genesand hasten their isolation. A similar approach could be used to isolateR genes effective against other diseases of woody plants. Furthermore,if the dspE operon is as widely conserved as is suggested by itshomology with the avrE locus, a corresponding R gene could be effectiveagainst a variety of pathogens both of woody and herbaceous plants.

Native (non-denatured) DspE protein has not been produced in sufficientquantity to test its ability to elicit the HR (i.e. hypersensitiveresponse) in a manner similar to hypersensitive response elicitors(i.e., by exogenous application). Therefore, no one has shown that dspEof E. amylovora elicits the HR when applied to plants as an isolatedcell-free material. However, when the gene encoding the protein istransferred to another bacterium (along with the smaller dspF gene),e.g., Pseudomonas syringae, which ordinarily causes disease on certainplants, the recipient bacterium no longer causes disease but insteadelicits the HR. The mechanism for this is not known for sure, but it issuspected to involve (and there is compelling evidence for) a mechanismin which the bacterial cell actually injects the DspE protein into theliving plant cell, triggering the development of plant cell collapse(i.e. HR). Presumably, when the DspE protein is in the living plantcell, it might signal the plant to develop resistance to insects andpathogens.

Based on the similarity of the predicted physical characteristics ofDspF to those of known chaperone proteins from animal pathogens, it isbelieved that this rather small protein is a chaperone of DspE.Chaperones in animal pathogens bind in the cytoplasm to specificproteins to be secreted. They seem to be required for secretion of theproteins but are not themselves secreted. Evidence suggests that thechaperones are not involved directly in targeting the secreted proteinsto the secretion apparatus. Instead, they may act to stabilize theproteins in the cytoplasm and/or prevent their premature aggregation orassociation with other proteins (e.g., bacterial proteins that directtransport through the host cell-membrane).

The dspE gene bears no similarity to known genes except avrE. Enzymaticfunction (i.e., one resulting in the production of a secondary moleculethat elicits the HR) of DspE cannot be ruled out at present. In fact,one avr gene product is known to elicit HR indirectly by catalyzingsynthesis of a diffusible elicitor molecule. However, the simplestexplanation for the observed HR eliciting function of the dspE operonexpressed in Pseudomonas species is that the protein encoded by the dspEgene is secreted from the bacterium and possibly transported into theplant cell, that there it triggers directly plant defense responsesleading to the HR, and that this process is mediated by a specificresistance gene product that recognizes (acts as a receptor of) the DspEprotein. Indeed, four avr genes that depend on the Hrp secretoryapparatus to function when expressed in bacteria have been shown tocause HR when expressed transgenically within plant cells. One of thesehas been shown to encode a protein that directly interacts with theproduct of its corresponding resistance gene. Ultimately, whether DspEelicits plant defense responses from outside or inside the plant cell,directly or through a secondary molecule, must be determined in order todefine practical applications of this protein and its encoding gene as aplant defense elicitor.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. An isolated hypersensitive response eliciting protein or polypeptideselected from the group consisting of a protein or polypeptide having anamino acid sequence comprising SEQ ID NO: 2 or SEQ ID NO: 4, and aprotein or polypeptide having an amino acid sequence encoded by anucleic acid whose full length complement hybridizes, at 65° C. in amedium which includes 1 M NaCl, to a DNA molecule comprising thenucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:
 3. 2. An isolatedprotein or polypeptide according to claim 1, wherein the protein orpolypeptide has an amino acid sequence comprising SEQ ID NO: 2 or SEQ IDNO:
 4. 3. An isolated protein or polypeptide according to claim 1,wherein the protein or polypeptide is encoded by a nucleic acid whosefull length complement hybridizes, at 65° C. in a medium which includes1 M NaCl, to a DNA molecule comprising the nucleotide sequence of SEQ IDNO: 1 or SEQ ID NO:
 3. 4. A method of imparting disease resistance to aplant comprising: applying a protein or polypeptide according claim 1 ina non-infectious form to a plant or plant seed under conditionseffective to impart disease resistance to the plant or a plant grownfrom the plant seed.
 5. A method according to claim 4, wherein saidapplying is carried out on a plant.
 6. A method according to claim 4,wherein said applying is carried out on a plant seed, said methodfurther comprising: planting the seed treated with the hypersensitiveresponse elicitor in natural or artificial soil and propagating a plantfrom the seed planted in the soil.
 7. A method of enhancing plant growthcomprising: applying a protein or polypeptide according claim 1 in anon-infectious form to a plant or plant seed under conditions effectiveto enhance growth of the plant or a plant grown from the plant seed. 8.A method according to claim 7, wherein said applying is carried out on aplant.
 9. A method according to claim 7, wherein said applying iscarried out on a plant seed, said method further comprising: plantingthe seed treated with the hypersensitive response elicitor in natural orartificial soil and propagating a plant from the seed planted in thesoil.
 10. A method of insect control for plants comprising: applying aprotein or polypeptide according claim 1 in a non-infectious form to aplant or plant seed under conditions effective to control insects on theplant or a plant grown from the plant seed.
 11. A method according toclaim 10, wherein said applying is carried out on a plant.
 12. A methodaccording to claim 10, wherein said applying is carried out on a plantseed, said method further comprising: planting the seed treated with thehypersensitive response elicitor in natural or artificial soil andpropagating a plant from the seed planted in the soil.
 13. A compositioncomprising: a protein or polypeptide according to claim 1 and a carrier.14. A composition according to claim 13 further comprising an additiveselected from the group consisting of fertilizer, insecticide,fungicide, nematacide, and mixtures thereof.